Pharmaceutical formulation for reducing frequency of urination and method of use thereof

ABSTRACT

Methods and compositions for reducing frequency of urination are disclosed. One method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising one or more analgesic agents.

This application is a continuation application of U.S. patentapplication Ser. No. 13/930,765, filed on Jun. 28, 2013, which is acontinuation-in-part application of U.S. patent application Ser. No.13/800,761, filed on Mar. 13, 2013. The entirety of the aforementionedapplications is incorporated herein by reference.

FIELD

The present application generally relates to methods and compositionsfor inhibiting the smooth muscles of the urinary bladder and, inparticular, to methods and compositions for reducing the frequency ofurination.

BACKGROUND

The detrusor muscle is a layer of the urinary bladder wall made ofsmooth muscle fibers arranged in spiral, longitudinal, and circularbundles. When the bladder is stretched, this signals the parasympatheticnervous system to contract the detrusor muscle. This encourages thebladder to expel urine through the urethra.

For the urine to exit the bladder, both the autonomically controlledinternal sphincter and the voluntarily controlled external sphinctermust be opened. Problems with these muscles can lead to incontinence. Ifthe amount of urine reaches 100% of the urinary bladder's absolutecapacity, the voluntary sphincter becomes involuntary and the urine willbe ejected instantly.

The human adult urinary bladder usually holds about 300-350 ml of urine(the working volume), but a full adult bladder may hold up to about 1000ml (the absolute volume), varying among individuals. As urineaccumulates, the ridges produced by folding of the wall of the bladder(rugae) flatten and the wall of the bladder thins as it stretches,allowing the bladder to store larger amounts of urine without asignificant rise in internal pressure.

In most individuals, the desire to urinate usually starts when thevolume of urine in the bladder reaches around 200 ml. At this stage itis easy for the subject, if desired, to resist the urge to urinate. Asthe bladder continues to fill, the desire to urinate becomes strongerand harder to ignore. Eventually, the bladder will fill to the pointwhere the urge to urinate becomes overwhelming, and the subject will nolonger be able to ignore it. In some individuals, this desire to urinatestarts when the bladder is less than 100% full in relation to itsworking volume. Such increased desire to urinate may interfere withnormal activities, including the ability to sleep for sufficientuninterrupted periods of rest. In some cases, this increased desire tourinate may be associated with medical conditions such as benignprostate hyperplasia or prostate cancer in men, or pregnancy in women.However, increased desire to urinate also occurs in individuals, bothmale and female, who are not affected by another medical condition.

Accordingly, there exists a need for compositions and methods for thetreatment of male and female subjects who suffer from a desire tourinate when the bladder is less than 100% full of urine in relation toits working volume. Said compositions and methods are needed for theinhibition of muscle contraction in order to allow in said subjects thedesire to urinate to start when the volume of urine in the bladderexceeds around 100% of its working volume.

SUMMARY

One aspect of the present application relates to a method for reducingfrequency of urination in a subject. The method comprises administeringto a subject in need thereof an effective amount of one or moreanalgesic agents and an effective amount of tadalafil.

Another aspect of the present application relates to a method forreducing frequency of urination in a subject. The method comprisesadministering to a subject in need thereof a pharmaceutical compositioncomprising an active ingredient comprising one or more analgesic agentsin an amount of 1-2000 mg per agent, and an inhibitor ofphosphodiesterase type 5 (PDE 5 inhibitor), wherein the one or moreanalgesic agents are selected from the group consisting of aspirin,ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, andacetaminophen.

Another aspect of the present application relates to a method forreducing the frequency of urination in a subject. The method comprisesadministering to a subject in need thereof a pharmaceutical compositioncomprising a first active ingredient comprising one or more analgesicagents and tadalafil, and a second active ingredient comprising one ormore agents selected from the group consisting of analgesic agents,antimuscarinic agents, antidiuretics, spasmolytics, PDE 5 inhibitors andzolpidem, wherein the first active ingredient is formulated forimmediate release and wherein the second active ingredient is formulatedfor extended release.

Another aspect of the present application relates to a pharmaceuticalcomposition comprising one or more analgesic agents, a PDE 5 inhibitorand a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are diagrams showing that analgesics regulate expressionof co-stimulatory molecules by Raw 264 macrophage cells in the absence(FIG. 1A) or presence (FIG. 1B) of LPS. Cells were cultures for 24 hrsin the presence of analgesic alone or together with Salmonellatyphimurium LPS (0.05 μg/ml). Results are mean relative % of CD40+CD80+cells.

DETAILED DESCRIPTION

The following detailed description is presented to enable any personskilled in the art to make and use the invention. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. The present invention is notintended to be limited to the embodiments shown, but is to be accordedthe broadest possible scope consistent with the principles and featuresdisclosed herein.

As used herein, the term “an effective amount” means an amount necessaryto achieve a selected result.

As used herein, the term “analgesic” refers to agents, compounds ordrugs used to relieve pain and inclusive of anti-inflammatory compounds.Exemplary analgesic and/or anti-inflammatory agents, compounds or drugsinclude, but are not limited to, non-steroidal anti-inflammatory drugs(NSAIDs), salicylates, aspirin, salicylic acid, methyl salicylate,diflunisal, salsalate, olsalazine, sulfasalazine, para-aminophenolderivatives, acetanilide, acetaminophen, phenacetin, fenamates,mefenamic acid, meclofenamate, sodium meclofenamate, heteroaryl aceticacid derivatives, tolmetin, ketorolac, diclofenac, propionic acidderivatives, ibuprofen, naproxen sodium, naproxen, fenoprofen,ketoprofen, flurbiprofen, oxaprozin; enolic acids, oxicam derivatives,piroxicam, meloxicam, tenoxicam, ampiroxicam, droxicam, pivoxicam,pyrazolon derivatives, phenylbutazone, oxyphenbutazone, antipyrine,aminopyrine, dipyrone, coxibs, celecoxib, rofecoxib, nabumetone,apazone, indomethacin, sulindac, etodolac, isobutylphenyl propionicacid, lumiracoxib, etoricoxib, parecoxib, valdecoxib, tiracoxib,etodolac, darbufelone, dexketoprofen, aceclofenac, licofelone,bromfenac, loxoprofen, pranoprofen, piroxicam, nimesulide, cizolirine,3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one,meloxicam, lornoxicam, d-indobufen, mofezolac, amtolmetin, pranoprofen,tolfenamic acid, flurbiprofen, suprofen, oxaprozin, zaltoprofen,alminoprofen, tiaprofenic acid, pharmacological salts thereof, hydratesthereof, and solvates thereof.

As used herein, the term “coxib” refers to a composition of compoundsthat is capable of inhibiting the activity or expression of COX2 enzymesor is capable of inhibiting or reducing the severity, including pain andswelling, of a severe inflammatory response.

As used herein, the term “derivative” refers to a chemically modifiedcompound wherein the modification is considered routine by the ordinaryskilled chemist, such as an ester or an amide of an acid, or protectinggroups such as a benzyl group for an alcohol or thiol, or atert-butoxycarbonyl group for an amine.

As used herein, the term “analogue” refers to a compound which comprisesa chemically modified form of a specific compound or class thereof andwhich maintains the pharmaceutical and/or pharmacological activitiescharacteristic of said compound or class.

As used herein, the term “pharmaceutically acceptable salts” refer toderivatives of the disclosed compounds wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include those derived frominorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, sulfamic acid, phosphoric acid, nitric acid, and the like and thesalts prepared from organic acids such as acetic acid, propionic acid,succinic acid, glycolic acid, stearic acid, lactic acid, malic acid,tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid,hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid,salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid,toluensulfonic acid, methanesulfonic acid, ethane dislfonic acid, oxalicacid, isethionic acid, and the like.

As used herein, the phrase “pharmaceutically acceptable” is used withreference to compounds, materials, compositions and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problems orcomplications commensurate with a reasonable benefit/risk ratio.

As used herein, the term “extended-release,” also known assustained-release (SR), sustained-action (SA), time-release (TR),controlled-release (CR), modified release (MR), or continuous-release(CR), refers to a mechanism used in medicine tablets or capsules todissolve slowly and release the active ingredient over time. Theadvantages of extended-release tablets or capsules are that they canoften be taken less frequently than immediate-release formulations ofthe same drug and that they keep steadier levels of the drug in thebloodstream, thus extending the duration of the drug action and loweringthe peak amount of drug in the bloodstream.

As used herein, the term “delayed-release” refers to a drug releaseprofile that the release of the active ingredient(s) of a pharmaceuticalcomposition is delayed or postponed for a given period of time (e.g.,the lag period) after administration of the pharmaceutical composition.

The term “immediate-release” is used herein with reference to a drugformulation that does not contain a dissolution rate controllingmaterial. There is substantially no delay in the release of the activeagents following administration of an immediate-release formulation. Animmediate-release coating may include suitable materials immediatelydissolving following administration so as to release the drug contentstherein. In some embodiments, the term “immediate-release” is used withreference to a drug formulation that release the active ingredientwithin two hours of administration.

One aspect of the present application relates to a method for reducingfrequency of urination by administering to a person in need thereof apharmaceutical composition. The pharmaceutical composition comprises oneor more analgesic agents, one or more inhibitors of phosphodiesterasetype 5 (PDE 5 inhibitors) and, optionally, one or more antimuscarinicagents, one or more antidiuretics and/or one or more spasmolytics. Thepharmaceutical composition may be formulated for immediate-release,extended-release, delayed-release, or combinations thereof.

In one embodiment, the pharmaceutical composition is formulated forextended-release by embedding the active ingredient in a matrix ofinsoluble substance(s) such as acrylics or chitin. An extended-releaseform is designed to release the analgesic compound at a predeterminedrate by maintaining a constant drug level for a specific period of time.This can be achieved through a variety of formulations, including, butnot limited to, liposomes and drug-polymer conjugates, such ashydrogels.

An extended-release formulation can be designed to release the activeagents at a predetermined rate so as to maintain a constant drug levelfor a specified, extended period of time, such as up to about 12 hours,about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about2 hours, or about 1 hour following administration or following a lagperiod associated with delayed-release of the drug.

In certain embodiments, the active agents are released over a timeinterval of between about 2 to about 12 hours. Alternatively, the activeagents may be released over about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10 hours, about 11 hours, or about 12 hours. Inyet other embodiments, the active agents are released over a time periodbetween about 5 to about 8 hours following administration.

In some embodiments, the extended-release formulation comprises anactive core comprised of one or more inert particles, each in the formof a bead, pellet, pill, granular particle, microcapsule, microsphere,microgranule, nanocapsule, or nanosphere coated on its surfaces withdrugs in the form of e.g., a drug-containing coating or film-formingcomposition using, for example, fluid bed techniques or othermethodologies known to those of skill in the art. The inert particle canbe of various sizes, so long as it is large enough to remain poorlydissolved. Alternatively, the active core may be prepared by granulatingand milling and/or by extrusion and spheronization of a polymercomposition containing the drug substance.

The active agents may be introduced to the inert carrier by techniquesknown to one skilled in the art, such as drug layering, powder coating,extrusion/spheronization, roller compaction or granulation. The amountof drug in the core will depend on the dose that is required andtypically varies from about 5 to 90 weight %. Generally, the polymericcoating on the active core will be from about 1 to 50% based on theweight of the coated particle, depending on the lag time required and/orthe polymers and coating solvents chosen. Those skilled in the art willbe able to select an appropriate amount of drug for coating onto orincorporating into the core to achieve the desired dosage. In oneembodiment, the inactive core may be a sugar sphere or a buffer crystalor an encapsulated buffer crystal such as calcium carbonate, sodiumbicarbonate, fumaric acid, tartaric acid, etc. which alters themicroenvironment of the drug to facilitate its release.

Extended-release formulations may utilize a variety of extended-releasecoatings or mechanisms facilitating the gradual release of active agentsover time. In some embodiments, the extended-release agent comprises apolymer controlling release by dissolution controlled release. In aparticular embodiment, the active agent(s) is incorporated in a matrixcomprising an insoluble polymer and drug particles or granules coatedwith polymeric materials of varying thickness. The polymeric materialmay comprise a lipid barrier comprising a waxy material, such ascarnauba wax, beeswax, spermaceti wax, candellila wax, shallac wax,cocoa butter, cetostearyl alcohol, partially hydrogenated vegetableoils, ceresin, paraffin wax, ceresine, myristyl alcohol, stearylalcohol, cetyl alcohol, and stearic acid, along with surfactants, suchas polyoxyethylene sorbitan monooleate. When contacted with an aqueousmedium, such as biological fluids, the polymer coating emulsifies orerodes after a predetermined lag-time depending on the thickness of thepolymer coating. The lag time is independent of gastrointestinalmotility, pH, or gastric residence.

In other embodiments, the extended-release agent comprises a polymericmatrix effecting diffusion controlled release. The matrix may compriseone or more hydrophilic and/or water-swellable, matrix forming polymers,pH-dependent polymers and/or pH-independent polymers.

In one embodiment, the extended-release formulation comprises a watersoluble or water-swellable matrix-forming polymer, optionally containingone or more solubility-enhancing agents and/or release-promoting agents.Upon solubilization of the water soluble polymer, the active agent(s)dissolves (if soluble) and gradually diffuses through the hydratedportion of the matrix. The gel layer grows with time as more waterpermeates into the core of the matrix, increasing the thickness of thegel layer and providing a diffusion barrier to drug release. As theouter layer becomes fully hydrated, the polymer chains become completelyrelaxed and can no longer maintain the integrity of the gel layer,leading to disentanglement and erosion of the outer hydrated polymer onthe surface of the matrix. Water continues to penetrate towards the corethrough the gel layer, until it has been completely eroded. Whereassoluble drugs are released by this combination of diffusion and erosionmechanisms, erosion is the predominant mechanism for insoluble drugs,regardless of dose.

Similarly, water-swellable polymers typically hydrate and swell inbiological fluids forming a homogenous matrix structure that maintainsits shape during drug release and serves as a carrier for the drug,solubility enhancers and/or release promoters. The initial matrixpolymer hydration phase results in slow-release of the drug (lag phase).Once the water swellable polymer is fully hydrated and swollen, waterwithin the matrix can similarly dissolve the drug substance and allowfor its diffusion out through the matrix coating.

Additionally, the porosity of the matrix can be increased due to theleaching out of pH-dependent release promoters so as to release the drugat a faster rate. The rate of the drug release then becomes constant andis a function of drug diffusion through the hydrated polymer gel. Therelease rate from the matrix is dependent upon various factors,including polymer type and level, drug solubility and dose, polymer todrug ratio, filler type and level, polymer to filler ratio, particlesize of drug and polymer, and porosity and shape of the matrix.

Exemplary hydrophilic and/or water-swellable, matrix forming polymersinclude, but are not limited to, cellulosic polymers includinghydroxyalkyl celluloses and carboxyalkyl celluloses such ashydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),hydroxyethylcellulose (HEC), methylcellulose (MC),carboxymethylcellulose (CMC); powdered cellulose such asmicrocrystalline cellulose, cellulose acetate, ethylcellulose, saltsthereof, and combinations thereof; alginates; gums includingheteropolysaccharide gums and homopolysaccharide gums such as xanthan,tragacanth, pectin, acacia, karaya, alginates, agar, guar, hydroxypropylguar, veegum, carrageenan, locust bean gum, gellan gum, and derivativestherefrom; acrylic resins including polymers and copolymers of acrylicacid, methacrylic acid, methyl acrylate, and methyl methacrylate; andcross-linked polyacrylic acid derivatives such as Carbomers (e.g.,CARBOPOL®, including CARBOPOL® 71G NF, which is available in variousmolecular weight grades from Noveon, Inc., Cincinnati, Ohio);carageenan; polyvinyl acetate (e.g., KOLLIDON® SR); and polyvinylpyrrolidone and its derivatives such as crospovidone, polyethyleneoxides, and polyvinyl alcohol. Preferred hydrophilic and water-swellablepolymers include the cellulosic polymers, especially HPMC.

The extended-release formulation may further comprise at least onebinder that is capable of cross-linking the hydrophilic compound to forma hydrophilic polymer matrix (i.e., a gel matrix) in an aqueous medium,including biological fluids.

Exemplary binders include homopolysaccharides such as galactomannangums, guar gum, hydroxypropyl guar gum, hydroxypropylcellulose (HPC;e.g., Klucel EXF), and locust bean gum. In other embodiments, the binderis an alginic acid derivative, HPC or microcrystallized cellulose (MCC).Other binders include, but are not limited to, starches,microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethylcellulose, hydroxypropylmethyl cellulose, and polyvinylpyrrolidone.

In one embodiment, the introduction method is drug layering by sprayinga suspension of active agent(s) and a binder onto the inert carrier.

The binder may be present in the bead formulation in an amount of fromabout 0.1% to about 15% by weight and preferably of from about 0.2% toabout 10% by weight.

In some embodiments, the hydrophilic polymer matrix may further includean ionic polymer, a non-ionic polymer, or water-insoluble hydrophobicpolymer to provide a stronger gel layer and/or reduce pore quantity anddimensions in the matrix so as to slow diffusion and erosion rates andconcomitant release of the active agent(s). This may additionallysuppress the initial burst effect and produce a more steady, “zero orderrelease” of active agent(s).

Exemplary ionic polymers for slowing dissolution rate include bothanionic and cationic polymers. Exemplary anionic polymers include, forexample, sodium carboxymethylcellulose (Na CMC); sodium alginate;polymers of acrylic acid or carbomers (e.g., CARBOPOL® 934, 940, 974PNF); enteric polymers such as polyvinyl acetate phthalate (PVAP),methacrylic acid copolymers (e.g., EUDRAGIT® L100, L 30D 55, A, and FS30D), and hypromellose acetate succinate (AQUAT HPMCAS); and xanthangum. Exemplary cationic polymers include, for example,dimethylaminoethyl methacrylate copolymer (e.g., EUDRAGIT® E 100).Incorporation of anionic polymers, particularly enteric polymers, isuseful for developing a pH-independent release profile for weakly basicdrugs as compared to hydrophilic polymer alone.

Exemplary non-ionic polymers for slowing dissolution rate, include, forexample, hydroxypropylcellulose (HPC) and polyethylene oxide (PEO)(e.g., POLYOX™)

Exemplary hydrophobic polymers include ethylcellulose (e.g., ETHOCEL™,SURELEASE®), cellulose acetate, methacrylic acid copolymers (e.g.,EUDRAGIT® NE 30D), ammonio-methacrylate copolymers (e.g., EUDRAGIT® RL100 or PO RS100), polyvinyl acetate, glyceryl monostearate, fatty acidssuch as acetyl tributyl citrate, and combinations and derivativesthereof.

The swellable polymer can be incorporated in the formulation inproportion from 1% to 50% by weight, preferably from 5% to 40% byweight, most preferably from 5% to 20% by weight. The swellable polymersand binders may be incorporated in the formulation either prior to orafter granulation. The polymers can also be dispersed in organicsolvents or hydro-alcohols and sprayed during granulation.

Exemplary release-promoting agents include pH-dependent enteric polymersthat remain intact at pH value lower than about 4.0 and dissolve at pHvalues higher than 4.0, preferably higher than 5.0, most preferablyabout 6.0, are considered useful as release-promoting agents for thisinvention. Exemplary pH-dependent polymers include, but are not limitedto, methacarylic acid copolymers; methacrylic acid-methyl methacrylatecopolymers (e.g., EUDRAGIT® L100 (Type A), EUDRAGIT® S100 (Type B), RohmGmbH, Germany); methacrylic acid-ethyl acrylate copolymers (e.g.,EUDRAGIT® L100-55 (Type C) and EUDRAGIT® L30D-55 copolymer dispersion,Rohm GmbH, Germany); copolymers of methacrylic acid-methyl methacrylateand methyl methacrylate (EUDRAGIT® FS); terpolymers of methacrylic acid,methacrylate, and ethyl acrylate; cellulose acetate phthalates (CAP);hydroxypropyl methylcellulose phthalate (HPMCP) (e.g., HP-55, HP-50,HP-55S, Shinetsu Chemical, Japan); polyvinyl acetate phthalates (PVAP)(e.g., COATERIC®, OPADRY® enteric white OY-P-7171); polyvinylbutyrateacetate; cellulose acetate succinates (CAS); hydroxypropylmethylcellulose acetate succinate (HPMCAS) (e.g., HPMCAS LF Grade, MFGrade, and HF Grade, including AQOAT® LF and AQOAT® MF, Shin-EtsuChemical, Japan); shellac (e.g., MARCOAT™ 125 and MARCOAT™ 125N); vinylacetate-maleic anhydride copolymer; styrene-maleic monoester copolymer;carboxymethyl ethylcellulose (CMEC, Freund Corporation, Japan);cellulose acetate phthalates (CAP) (e.g., AQUATERIC®); cellulose acetatetrimellitates (CAT); and mixtures of two or more thereof at weightratios between about 2:1 to about 5:1, such as a mixture of EUDRAGIT® L100-55 and EUDRAGIT® S 100 at a weight ratio of about 3:1 to about 2:1or a mixture of EUDRAGIT® L 30 D-55 and EUDRAGIT® FS at a weight ratioof about 3:1 to about 5:1.

These polymers may be used either alone or in combination, or togetherwith polymers other than those mentioned above. Preferred entericpH-dependent polymers are the pharmaceutically acceptable methacrylicacid copolymers. These copolymers are anionic polymers based onmethacrylic acid and methyl methacrylate and, preferably, have a meanmolecular weight of about 135,000. A ratio of free carboxyl groups tomethyl-esterified carboxyl groups in these copolymers may range, forexample, from 1:1 to 1:3, e.g. around 1:1 or 1:2. Such polymers are soldunder the trade name Eudragit® such as the Eudragit L series e.g.,Eudragit L 12.5®, Eudragit L 12.5P®, Eudragit L100®, Eudragit L 100-55®,Eudragit L-30D®, Eudragit L-30 D-55®, the Eudragit S® series e.g.,Eudragit S 12.5®, Eudragit S 12.5P®, Eudragit 5100°. The releasepromoters are not limited to pH dependent polymers. Other hydrophilicmolecules that dissolve rapidly and leach out of the dosage form quicklyleaving a porous structure can be also be used for the same purpose.

In some embodiments, the matrix may include a combination of releasepromoters and solubility enhancing agents. The solubility enhancingagents can be ionic and non-ionic surfactants, complexing agents,hydrophilic polymers, and pH modifiers such as acidifying agents andalkalinizing agents, as well as molecules that increase the solubilityof poorly soluble drug through molecular entrapment. Several solubilityenhancing agents can be utilized simultaneously.

Solubility enhancing agents may include surface active agents, such assodium docusate; sodium lauryl sulfate; sodium stearyl fumarate; Tweens®and Spans (PEO modified sorbitan monoesters and fatty acid sorbitanesters); poly(ethylene oxide)-polypropylene oxide-poly(ethylene oxide)block copolymers (aka PLURONICS™); complexing agents such as lowmolecular weight polyvinyl pyrrolidone and low molecular weighthydroxypropyl methyl cellulose; molecules that aid solubility bymolecular entrapment such as cyclodextrins and pH modifying agents,including acidifying agents such as citric acid, fumaric acid, tartaricacid, and hydrochloric acid; and alkalizing agents such as meglumine andsodium hydroxide.

Solubility enhancing agents typically constitute from 1% to 80% byweight, preferably from 1% to 60%, more preferably from 1% to 50%, ofthe dosage form and can be incorporated in a variety of ways. They canbe incorporated in the formulation prior to granulation in dry or wetform. They can also be added to the formulation after the rest of thematerials are granulated or otherwise processed. During granulation,solubility enhancing agents can be sprayed as solutions with or withouta binder.

In one embodiment, the extended-release formulation comprises awater-insoluble water-permeable polymeric coating or matrix comprisingone or more water-insoluble water-permeable film-forming over the activecore. The coating may additionally include one or more water solublepolymers and/or one or more plasticizers. The water-insoluble polymercoating comprises a barrier coating for release of active agents in thecore, wherein lower molecular weight (viscosity) grades exhibit fasterrelease rates as compared to higher viscosity grades.

In some embodiments, the water-insoluble film-forming polymers includeone or more alkyl cellulose ethers, such as ethyl celluloses andmixtures thereof, (e.g., ethyl cellulose grades PR100, PR45, PR20, PR10,and PR7; ETHOCEL®, Dow).

In some embodiments, the water-insoluble polymer provides suitableproperties (e.g., extended-release characteristics, mechanicalproperties, and coating properties) without the need for a plasticizer.For example, coatings comprising polyvinyl acetate (PVA), neutralcopolymers of acrylate/methacrylate esters such as commerciallyavailable Eudragit NE30D from Evonik Industries, ethyl cellulose incombination with hydroxypropylcellulose, waxes, etc. can be appliedwithout plasticizers.

In yet another embodiment, the water-insoluble polymer matrix mayfurther include a plasticizer. The amount of plasticizer requireddepends upon the plasticizer, the properties of the water-insolublepolymer and the ultimate desired properties of the coating. Suitablelevels of plasticizer range from about 1% to about 20%, from about 3% toabout 20%, about 3% to about 5%, about 7% to about 10%, about 12% toabout 15%, about 17% to about 20%, or about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 15%, or about 20% by weight relative to the total weight of thecoating, inclusive of all ranges and sub-ranges therebetween.

Exemplary plasticizers include, but are not limited to, triacetin,acetylated monoglyceride, oils (castor oil, hydrogenated castor oil,grape seed oil, sesame oil, olive oil, and etc.), citrate esters,triethyl citrate, acetyltriethyl citrate acetyltributyl citrate,tributyl citrate, acetyl tri-n-butyl citrate, diethyl phthalate, dibutylphthalate, dioctyl phthalate, methyl paraben, propyl paraben, propylparaben, butyl paraben, diethyl sebacate, dibutyl sebacate,glyceroltributyrate, substituted triglycerides and glycerides,monoacetylated and diacetylated glycerides (e.g., MYVACET® 9-45),glyceryl monostearate, glycerol tributyrate, polysorbate 80,polyethyleneglycol (such as PEG-4000 and PEG-400), propyleneglycol,1,2-propyleneglycol, glycerin, sorbitol, diethyl oxalate, diethylmalate, diethyl fumarate, diethylmalonate, dibutyl succinate, fattyacids, glycerin, sorbitol, diethyl oxalate, diethyl malate, diethylmaleate, diethyl fumarate, diethyl succinate, diethyl malonate, dioctylphthalate, dibutyl sebacate, and mixtures thereof. The plasticizer canhave surfactant properties, such that it can act as a release modifier.For example, non-ionic detergents such as Brij 58 (polyoxyethylene (20)cetyl ether), and the like, can be used.

Plasticizers can be high boiling point organic solvents used to impartflexibility to otherwise hard or brittle polymeric materials and canaffect the release profile for the active agent(s). Plasticizersgenerally cause a reduction in the cohesive intermolecular forces alongthe polymer chains resulting in various changes in polymer propertiesincluding a reduction in tensile strength and increase in elongation anda reduction in the glass transition or softening temperature of thepolymer. The amount and choice of the plasticizer can affect thehardness of a tablet, for example, and can even affect its dissolutionor disintegration characteristics, as well as its physical and chemicalstability. Certain plasticizers can increase the elasticity and/orpliability of a coat, thereby decreasing the coat's brittleness.

In another embodiment, the extended-release formulation comprises acombination of at least two gel-forming polymers, including at least onenon-ionic gel-forming polymer and/or at least one anionic gel-formingpolymer. The gel formed by the combination of gel-forming polymersprovides controlled release, such that when the formulation is ingestedand comes into contact with the gastrointestinal fluids, the polymersnearest the surface hydrate to form a viscous gel layer. Because of thehigh viscosity, the viscous layer dissolves away only gradually,exposing the material below to the same process. The mass thus dissolvesaway slowly, thereby slowly releasing the active ingredient into thegastrointestinal fluids. The combination of at least two gel-formingpolymers enables properties of the resultant gel, such as viscosity, tobe manipulated in order to provide the desired release profile.

In a particular embodiment, the formulation comprises at least onenon-ionic gel-forming polymer and at least one anionic gel-formingpolymer. In another embodiment, the formulation comprises two differentnon-ionic gel-forming polymers. In yet another embodiment, theformulation comprises a combination of non-ionic gel-forming polymerswith the same chemistry, but solubilities, viscosities, and/or molecularweights (for example a combination of hydroxyproplyl methylcellulose ofdifferent viscosity grades, such as HPMC K100 and HPMC K15M or HPMCK100M).

Exemplary anionic gel forming polymers include, but are not limited to,sodium carboxymethylcellulose (Na CMC), carboxymethyl cellulose (CMC),anionic polysaccharides such as sodium alginate, alginic acid, pectin,polyglucuronic acid (poly-α- and -β-1,4-glucuronic acid),polygalacturonic acid (pectic acid), chondroitin sulfate, carrageenan,furcellaran, anionic gums such as xanthan gum, polymers of acrylic acidor carbomers (Carbopol® 934, 940, 974P NF), Carbopol® copolymers, aPemulen® polymer, polycarbophil, and others.

Exemplary non-ionic gel-forming polymers include, but are not limitedto, Povidone (PVP: polyvinyl pyrrolidone), polyvinyl alcohol, copolymerof PVP and polyvinyl acetate, HPC (hydroxypropyl cellulose), HPMC(hydroxypropyl methylcellulose), hydroxyethyl cellulose, hydroxymethylcellulose, gelatin, polyethylene oxide, acacia, dextrin, starch,polyhydroxyethylmethacrylate (PHEMA), water soluble nonionicpolymethacrylates and their copolymers, modified cellulose, modifiedpolysaccharides, nonionic gums, nonionic polysaccharides, and/ormixtures thereof.

The formulation may optionally comprise an enteric polymer as describedabove and/or at least one excipient, such as a filler, a binder (asdescribed above), a disintegrant and/or a flow aid or glidant.

Exemplary fillers include but are not limited to, lactose; glucose;fructose; sucrose; dicalcium phosphate; sugar alcohols also known as“sugar polyol” such as sorbitol, manitol, lactitol, xylitol, isomalt,erythritol, and hydrogenated starch hydrolysates (a blend of severalsugar alcohols); corn starch; potato starch; sodiumcarboxymethycellulose; ethylcellulose and cellulose acetate; entericpolymers; or a mixture thereof.

Exemplary binders include, but are not limited to, water-solublehydrophilic polymers such as Povidone (PVP: polyvinyl pyrrolidone),copovidone (a copolymer of polyvinyl pyrrolidone and polyvinyl acetate),low molecular weight HPC (hydroxypropyl cellulose), low molecular weightHPMC (hydroxypropyl methylcellulose), low molecular weight carboxymethyl cellulose, ethylcellulose, gelatin, polyethylene oxide, acacia,dextrin, magnesium aluminum silicate, and starch and polymethacrylatessuch as Eudragit NE 30D, Eudragit RL, Eudragit RS, Eudragit E, polyvinylacetate, enteric polymers, or mixtures thereof.

Exemplary disintegrants include, but are not limited to, low-substitutedcarboxymethyl cellulose sodium, crospovidone (cross-linked polyvinylpyrrolidone), sodium carboxymethyl starch (sodium starch glycolate),cross-linked sodium carboxymethyl cellulose (Croscarmellose),pregelatinized starch (starch 1500), microcrystalline cellulose, waterinsoluble starch, calcium carboxymethyl cellulose, low substitutedhydroxypropyl cellulose, and magnesium or aluminum silicate.

Exemplary glidants include but are not limited to, magnesium, silicondioxide, talc, starch, titanium dioxide, and the like.

In yet another embodiment, the extended-release formulation is formed bycoating a water soluble/dispersible drug-containing particle, such as abead or bead population therein (as described above), with a coatingmaterial and, optionally, a pore former and other excipients. Thecoating material is preferably selected from a group comprisingcellulosic polymers such as ethylcellulose (e.g., SURELEASE®),methylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose,cellulose acetate, and cellulose acetate phthalate; polyvinyl alcohol;acrylic polymers such as polyacrylates, polymethacrylates, andcopolymers thereof and other water-based or solvent-based coatingmaterials. The release-controlling coating for a given bead populationmay be controlled by at least one parameter of the release controllingcoating, such as the nature of the coating, coating level, type andconcentration of a pore former, process parameters, and combinationsthereof. Thus, changing a parameter, such as a pore formerconcentration, or the conditions of the curing, allows for changes inthe release of active agent(s) from any given bead population, therebyallowing for selective adjustment of the formulation to a pre-determinedrelease profile.

Pore formers suitable for use in the release controlling coating hereincan be organic or inorganic agents and include materials that can bedissolved, extracted or leached from the coating in the environment ofuse. Exemplary pore forming agents include, but are not limited to,organic compounds such as mono-, oligo-, and polysaccharides includingsucrose, glucose, fructose, mannitol, mannose, galactose, sorbitol,pullulan, and dextran; polymers soluble in the environment of use suchas water-soluble hydrophilic polymers, hydroxyalkylcelluloses,carboxyalkylcelluloses, hydroxypropylmethylcellulose, cellulose ethers,acrylic resins, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone,polyethylene oxide, Carbowaxes, Carbopol, and the like, diols, polyols,polyhydric alcohols, polyalkylene glycols, polyethylene glycols,polypropylene glycols, or block polymers thereof, polyglycols, andpoly(α-Ω)alkylenediols; and inorganic compounds such as alkali metalsalts, lithium carbonate, sodium chloride, sodium bromide, potassiumchloride, potassium sulfate, potassium phosphate, sodium acetate, sodiumcitrate, suitable calcium salts, combination thereof, and the like.

The release controlling coating can further comprise other additivesknown in the art, such as plasticizers, anti-adherents, glidants (orflow aids), and antifoams.

In some embodiments, the coated particles or beads may additionallyinclude an “overcoat,” to provide, e.g., moisture protection, staticcharge reduction, taste-masking, flavoring, coloring, and/or polish orother cosmetic appeal to the beads. Suitable coating materials for suchan overcoat are known in the art and include, but are not limited to,cellulosic polymers such as hydroxypropylmethylcellulose,hydroxypropylcellulose, and microcrystalline cellulose or combinationsthereof (for example, various OPADRY® coating materials).

The coated particles or beads may additionally contain enhancers thatmay be exemplified by, but not limited to, solubility enhancers,dissolution enhancers, absorption enhancers, permeability enhancers,stabilizers, complexing agents, enzyme inhibitors, p-glycoproteininhibitors, and multidrug resistance protein inhibitors. Alternatively,the formulation can also contain enhancers that are separated from thecoated particles, for example in a separate population of beads or as apowder. In yet another embodiment, the enhancer(s) may be contained in aseparate layer on coated particles either under or above the releasecontrolling coating.

In other embodiments, the extended-release formulation is formulated torelease the active agent(s) by an osmotic mechanism. By way of example,a capsule may be formulated with a single osmotic unit or it mayincorporate 2, 3, 4, 5, or 6 push-pull units encapsulated within a hardgelatin capsule, whereby each bilayer push pull unit contains an osmoticpush layer and a drug layer, both surrounded by a semi-permeablemembrane. One or more orifices are drilled through the membrane next tothe drug layer. This membrane may be additionally covered with apH-dependent enteric coating to prevent release until after gastricemptying. The gelatin capsule dissolves immediately after ingestion. Asthe push pull unit(s) enters the small intestine, the enteric coatingbreaks down, which then allows fluid to flow through the semi-permeablemembrane, swelling the osmotic push compartment to force to force drugsout through the orifice(s) at a rate precisely controlled by the rate ofwater transport through the semi-permeable membrane. Release of drugscan occur over a constant rate for up to 24 hours or more.

The osmotic push layer comprises one or more osmotic agents creating thedriving force for transport of water through the semi-permeable membraneinto the core of the delivery vehicle. One class of osmotic agentsincludes water-swellable hydrophilic polymers, also referred to as“osmopolymers” and “hydrogels,” including, but not limited to,hydrophilic vinyl and acrylic polymers, polysaccharides such as calciumalginate, polyethylene oxide (PEO), polyethylene glycol (PEG),polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate),poly(acrylic) acid, poly(methacrylic) acid, polyvinylpyrrolidone (PVP),crosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers, PVA/PVPcopolymers with hydrophobic monomers such as methyl methacrylate andvinyl acetate, hydrophilic polyurethanes containing large PEO blocks,sodium croscarmellose, carrageenan, hydroxyethyl cellulose (HEC),hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC),carboxymethyl cellulose (CMC) and carboxyethyl, cellulose (CEC), sodiumalginate, polycarbophil, gelatin, xanthan gum, and sodium starchglycolate.

Another class of osmotic agents includes osmogens, which are capable ofimbibing water to effect an osmotic pressure gradient across thesemi-permeable membrane. Exemplary osmogens include, but are not limitedto, inorganic salts such as magnesium sulfate, magnesium chloride,calcium chloride, sodium chloride, lithium chloride, potassium sulfate,potassium phosphates, sodium carbonate, sodium sulfite, lithium sulfate,potassium chloride, and sodium sulfate; sugars such as dextrose,fructose, glucose, inositol, lactose, maltose, mannitol, raffinose,sorbitol, sucrose, trehalose, and xylitol; organic acids such asascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid,sebacic acid, sorbic acid, adipic acid, edetic acid, glutamic acid,p-toluenesulfonic acid, succinic acid, and tartaric acid; urea; andmixtures thereof.

Materials useful in forming the semipermeable membrane include variousgrades of acrylics, vinyls, ethers, polyamides, polyesters, andcellulosic derivatives that are water-permeable and water-insoluble atphysiologically relevant pHs, or are susceptible to being renderedwater-insoluble by chemical alteration, such as crosslinking.

In some embodiments, the extended-release formulation comprises apolysaccharide coating that is resistant to erosion in both the stomachand intestine. Such polymers can be only degraded in the colon, whichcontains a large microflora containing biodegradable enzymes breakingdown, for example, the polysaccharide coatings to release the drugcontents in a controlled, time-dependent manner. Exemplarypolysaccharide coatings may include, for example, amylose,arabinogalactan, chitosan, chondroitin sulfate, cyclodextrin, dextran,guar gum, pectin, xylan, and combinations or derivatives therefrom.

In some embodiments, the pharmaceutical composition is formulated fordelayed extended-release. As used herein, the term “delayedextended-release” is used with reference to a drug formulation having arelease profile in which there is a predetermined delay in the releaseof the drug following administration and, once initiated, the drug isreleased continuously over an extended period of time. In someembodiments, the delayed extended-release formulation includes anextended-release formulation coated with an enteric coating, which is abarrier applied to oral medication that prevents release of medicationbefore it reaches the small intestine. Delayed-release formulations,such as enteric coatings, prevent drugs having an irritant effect on thestomach, such as aspirin, from dissolving in the stomach. Such coatingsare also used to protect acid-unstable drugs from the stomach's acidicexposure, delivering them instead to a basic pH environment (intestine'spH 5.5 and above) where they do not degrade and give their desiredaction. The term “pulsatile release” is a type of delayed-release, whichis used herein with reference to a drug formulation that provides rapidand transient release of the drug within a short time period immediatelyafter a predetermined lag period, thereby producing a “pulsed” plasmaprofile of the drug after drug administration. Formulations may bedesigned to provide a single pulsatile release or multiple pulsatilereleases at predetermined time intervals following administration, or apulsatile release (e.g., 20-60% of the active ingredient) followed withextended release over a period of time (e.g., a continuous release ofthe remainder of the active ingredient). A delayed-release or pulsatilerelease formulation generally comprises one or more elements coveredwith a barrier coating, which dissolves, erodes or ruptures following aspecified lag phase.

A barrier coating for delayed-release may consist of a variety ofdifferent materials, depending on the objective. In addition, aformulation may comprise a plurality of barrier coatings to facilitaterelease in a temporal manner. The coating may be a sugar coating, a filmcoating (e.g., based on hydroxypropyl methylcellulose, methylcellulose,methyl hydroxyethylcellulose, hydroxypropylcellulose,carboxymethylcellulose, acrylate copolymers, polyethylene glycols,and/or polyvinylpyrrolidone) or a coating based on methacrylic acidcopolymer, cellulose acetate phthalate, hydroxypropyl methylcellulosephthalate, hydroxypropyl methylcellulose acetate succinate, polyvinylacetate phthalate, shellac, and/or ethylcellulose. Furthermore, theformulation may additionally include a time delay material such as, forexample, glyceryl monostearate or glyceryl distearate.

In some embodiments, the delayed, extended-release formulation includesan enteric coating comprised one or more polymers facilitating releaseof active agents in proximal or distal regions of the gastrointestinaltract. As used herein, the term “enteric coating” is a coatingcomprising of one or more polymers having a pH dependent orpH-independent release profile. An enteric coated pill will not dissolvein the acidic juices of the stomach (pH ˜3), but they will in thealkaline (pH 7-9) environment present in the small intestine or colon.An enteric polymer coating typically resists releases of the activeagents until some time after a gastric emptying lag period of about 3-4hours after administration.

pH dependent enteric coatings comprise one or more pH-dependent orpH-sensitive polymers that maintain their structural integrity at lowpH, as in the stomach, but dissolve in higher pH environments in moredistal regions of the gastrointestinal tract, such as the smallintestine, where the drug contents are released. For purposes of thepresent invention, “pH dependent” is defined as having characteristics(e.g., dissolution) which vary according to environmental pH. ExemplarypH-dependent polymers have been described earlier. pH-dependent polymerstypically exhibit a characteristic pH optimum for dissolution. In someembodiments, the pH-dependent polymer exhibits a pH optimum betweenabout 5.0 and 5.5, between about 5.5 and 6.0, between about 6.0 and 6.5,or between about 6.5 and 7.0. In other embodiments, the pH-dependentpolymer exhibits a pH optimum of ≧5.0, of ≧5.5, of ≧6.0, of ≧6.5, or of≧7.0.

In certain embodiments, the coating methodology employs the blending ofone or more pH-dependent and one or more pH-independent polymers. Theblending of pH-dependent and pH-independent polymers can reduce therelease rate of active ingredients once the soluble polymer has reachedits optimum pH of solubilization.

In some embodiments, a “time-controlled” or “time-dependent” releaseprofile can be obtained using a water insoluble capsule body containingone or more active agents, wherein the capsule body closed at one endwith an insoluble, but permeable and swellable hydrogel plug. Uponcontact with gastrointestinal fluid or dissolution medium, the plugswells, pushing itself out of the capsule and releasing the drugs aftera pre-determined lag time, which can be controlled by e.g., the positionand dimensions of the plug. The capsule body may be further coated withan outer pH-dependent enteric coating keeping the capsule intact untilit reaches the small intestine. Suitable plug materials include, forexample, polymethacrylates, erodible compressed polymers (e.g., HPMC,polyvinyl alcohol), congealed melted polymer (e.g., glyceryl monooleate), and enzymatically controlled erodible polymers (e.g.,polysaccharides, such as amylose, arabinogalactan, chitosan, chondroitinsulfate, cyclodextrin, dextran, guar gum, pectin and xylan).

In other embodiments, capsules or bilayered tablets may be formulated tocontain a drug-containing core, covered by a swelling layer and an outerinsoluble, but semi-permeable polymer coating or membrane. The lag timeprior to rupture can be controlled by the permeation and mechanicalproperties of the polymer coating and the swelling behavior of theswelling layer. Typically, the swelling layer comprises one or moreswelling agents, such as swellable hydrophilic polymers that swell andretain water in their structures.

Exemplary water swellable materials to be used in the delayed-releasecoating include, but are not limited to, polyethylene oxides (havinge.g., an average molecular weight between 1,000,000 and 7,000,000, suchas POLYOX®); methylcellulose; hydroxypropyl cellulose; hydroxypropylmethylcellulose; polyalkylene oxides having a weight average molecularweight of 100,000 to 6,000,000, including, but not limited to,poly(methylene oxide), poly(butylene oxide), poly(hydroxy alkylmethacrylate) having a molecular weight of 25,000 to 5,000,000,poly(vinyl)alcohol having a low acetal residue, which is cross-linkedwith glyoxal, formaldehyde, or glutaraldehyde, and having a degree ofpolymerization from 200 to 30,000; mixtures of methyl cellulose,cross-linked agar, and carboxymethyl cellulose; hydrogel formingcopolymers produced by forming a dispersion of a finely dividedcopolymer of maleic anhydride with styrene, ethylene, propylene,butylene, or isobutylene cross-linked with from 0.001 to 0.5 moles ofsaturated cross-linking agent per mole of maleic anyhydride in thecopolymer; CARBOPOL® acidic carboxy polymers having a molecular weightof 450,000 to 4,000,000; CYANAMER® polyacrylamides; cross-linked waterswellable indenemaleicanhydride polymers; GOODRITE® polyacrylic acidhaving a molecular weight of 80,000 to 200,000; starch graft copolymers;AQUA-KEEPS® acrylate polymer polysaccharides composed of condensedglucose units such as diester cross-linked polyglucan; carbomers havinga viscosity of 3,000 to 60,000 mPa as a 0.5%-1% w/v aqueous solution;cellulose ethers such as hydroxypropylcellulose having a viscosity ofabout 1000-7000 mPa s as a 1% w/w aqueous solution (25° C.);hydroxypropyl methylcellulose having a viscosity of about 1000 orhigher, preferably 2,500 or higher to a maximum of 25,000 mPa as a 2%w/v aqueous solution; polyvinylpyrrolidone having a viscosity of about300-700 mPa s as a 10% w/v aqueous solution at 20° C.; and combinationsthereof.

Alternatively, the release time of the drugs can be controlled by adisintegration lag time depending on the balance between thetolerability and thickness of a water insoluble polymer membrane (suchas ethyl cellulose, EC) containing predefined micropores at the bottomof the body and the amount of a swellable excipient, such as lowsubstituted hydroxypropyl cellulose (L-HPC) and sodium glycolate. Afteroral administration, GI fluids permeate through the micropores, causingswelling of the swellable excipients, which produces an inner pressuredisengaging the capsular components, including a first capsule bodycontaining the swellable materials, a second capsule body containing thedrugs, and an outer cap attached to the first capsule body.

The enteric layer may further comprise anti-tackiness agents, such astalc or glyceryl monostearate and/or plasticizers. The enteric layer mayfurther comprise one or more plasticizers including, but not limited to,triethyl citrate, acetyl triethyl citrate, acetyltributyl citrate,polyethylene glycol acetylated monoglycerides, glycerin, triacetin,propylene glycol, phthalate esters (e.g., diethyl phthalate, dibutylphthalate), titanium dioxide, ferric oxides, castor oil, sorbitol, anddibutyl sebacate.

In another embodiment, the delayed release formulation employs awater-permeable but insoluble film coating to enclose the activeingredient and an osmotic agent. As water from the gut slowly diffusesthrough the film into the core, the core swells until the film bursts,thereby releasing the active ingredients. The film coating may beadjusted to permit various rates of water permeation or release time.

In another embodiment, the delayed release formulation employs awater-impermeable tablet coating whereby water enters through acontrolled aperture in the coating until the core bursts. When thetablet bursts, the drug contents are released immediately or over alonger period of time. These and other techniques may be modified toallow for a pre-determined lag period before release of drugs isinitiated.

In another embodiment, the active agents are delivered in a formulationto provide both delayed-release and extended-release(delayed-extended-release). The term “delayed-extended-release” is usedherein with reference to a drug formulation providing pulsatile releaseof active agents at a pre-determined time or lag period followingadministration, which is then followed by extended-release of the activeagents thereafter.

In some embodiments, immediate-release, extended-release,delayed-release, or delayed-extended-release formulations comprises anactive core comprised of one or more inert particles, each in the formof a bead, pellet, pill, granular particle, microcapsule, microsphere,microgranule, nanocapsule, or nanosphere coated on its surfaces withdrugs in the form of e.g., a drug-containing film-forming compositionusing, for example, fluid bed techniques or other methodologies known tothose of skill in the art. The inert particle can be of various sizes,so long as it is large enough to remain poorly dissolved. Alternatively,the active core may be prepared by granulating and milling and/or byextrusion and spheronization of a polymer composition containing thedrug substance.

The amount of drug in the core will depend on the dose that is requiredand typically varies from about 5 to 90 weight %. Generally, thepolymeric coating on the active core will be from about 1 to 50% basedon the weight of the coated particle, depending on the lag time and typeof release profile required and/or the polymers and coating solventschosen. Those skilled in the art will be able to select an appropriateamount of drug for coating onto or incorporating into the core toachieve the desired dosage. In one embodiment, the inactive core may bea sugar sphere or a buffer crystal or an encapsulated buffer crystalsuch as calcium carbonate, sodium bicarbonate, fumaric acid, tartaricacid, etc. which alters the microenvironment of the drug to facilitateits release.

In some embodiments, for example, delayed-release ordelayed-extended-release compositions may formed by coating a watersoluble/dispersible drug-containing particle, such as a bead, with amixture of a water insoluble polymer and an enteric polymer, wherein thewater insoluble polymer and the enteric polymer may be present at aweight ratio of from 4:1 to 1:1, and the total weight of the coatings is10 to 60 weight % based on the total weight of the coated beads. Thedrug layered beads may optionally include an inner dissolution ratecontrolling membrane of ethylcellulose. The composition of the outerlayer, as well as the individual weights of the inner and outer layersof the polymeric membrane are optimized for achieving desired circadianrhythm release profiles for a given active, which are predicted based onin vitro/in vivo correlations.

In other embodiments the formulations may comprise a mixture ofimmediate-release drug-containing particles without a dissolution ratecontrolling polymer membrane and delayed-extended-release beadsexhibiting, for example, a lag time of 2-4 hours following oraladministration, thus providing a two-pulse release profile.

In some embodiments, the active core is coated with one or more layersof dissolution rate-controlling polymers to obtain desired releaseprofiles with or without a lag time. An inner layer membrane can largelycontrol the rate of drug release following imbibition of water or bodyfluids into the core, while the outer layer membrane can provide for adesired lag time (the period of no or little drug release followingimbibition of water or body fluids into the core). The inner layermembrane may comprise a water insoluble polymer, or a mixture of waterinsoluble and water soluble polymers.

The polymers suitable for the outer membrane, which largely controls thelag time of up to 6 hours may comprise an enteric polymer, as describedabove, and a water insoluble polymer at 10 to 50 weight %. The ratio ofwater insoluble polymer to enteric polymer may vary from 4:1 to 1:2,preferably the polymers are present at a ratio of about 1:1. The waterinsoluble polymer typically used is ethylcellulose.

Exemplary water insoluble polymers include ethylcellulose, polyvinylacetate (Kollicoat SR#0D from BASF), neutral copolymers based on ethylacrylate and methylmethacrylate, copolymers of acrylic and methacrylicacid esters with quaternary ammonium groups such as EUDRAGIT® NE, RS andRS30D, RL or RL30D, and the like. Exemplary water soluble polymersinclude low molecular weight HPMC, HPC, methylcellulose, polyethyleneglycol (PEG of molecular weight>3000) at a thickness ranging from 1weight % up to 10 weight % depending on the solubility of the active inwater and the solvent or latex suspension based coating formulationused. The water insoluble polymer to water soluble polymer may typicallyvary from 95:5 to 60:40, preferably from 80:20 to 65:35. In someembodiments, AMBERLITE™ IRP69 resin is used as an extended-releasecarrier. AMBERLITE™ IRP69 is an insoluble, strongly acidic, sodium formcation exchange resin that is suitable as carrier for cationic (basic)substances. In other embodiments, DUOLITE™ AP143/1093 resin is used asan extended-release carrier. DUOLITE™ AP143/1093 is an insoluble,strongly basic, anion exchange resin that is suitable as a carrier foranionic (acidic) substances. When used as a drug carrier, AMBERLITE™IRP69 or/and DUOLITE™ AP143/1093 resin provides a means for bindingmedicinal agents onto an insoluble polymeric matrix. Extended-release isachieved through the formation of resin-drug complexes (drug resinates).The drug is released from the resin in vivo as the drug reachesequilibrium with the high electrolyte concentrations, which are typicalof the gastrointestinal tract. More hydrophobic drugs will usually elutefrom the resin at a lower rate, owing to hydrophobic interactions withthe aromatic structure of the cation exchange system.

In some embodiments, the pharmaceutical composition is formulated fororal administration. Oral dosage forms include, for example, tablets,capsules, and caplets and may also comprise a plurality of granules,beads, powders, or pellets that may or may not be encapsulated. Tabletsand capsules represent the most convenient oral dosage forms, in whichcase solid pharmaceutical carriers are employed.

In a delayed-release formulation, one or more barrier coatings may beapplied to pellets, tablets, or capsules to facilitate slow dissolutionand concomitant release of drugs into the intestine. Typically, thebarrier coating contains one or more polymers encasing, surrounding, orforming a layer, or membrane around the therapeutic composition oractive core. In some embodiments, the active agents are delivered in aformulation to provide delayed-release at a pre-determined timefollowing administration. The delay may be up to about 10 minutes, about20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3hours, about 4 hours, about 5 hours, about 6 hours, or longer.

Various coating techniques may be applied to granules, beads, powders orpellets, tablets, capsules or combinations thereof containing activeagents to produce different and distinct release profiles. In someembodiments, the pharmaceutical composition is in a tablet or capsuleform containing a single coating layer. In other embodiments, thepharmaceutical composition is in a tablet or capsule form containingmultiple coating layers. In some embodiments, the pharmaceuticalcomposition of the present application is formulated forextended-release or delayed extended-release of 100% of the activeingredient.

In other embodiments, the pharmaceutical composition of the presentapplication is formulated for a two-phase extended-release or delayedtwo-phase extended-release characterized by an “immediate-release”component that is released within two hours of administration and an“extended-release” component which is released over a period of 2-12hours. In some embodiments, the “immediate-release” component providesabout 20-60% of the total dosage of the active agent(s) and the“extended-release” component provides 40-80% of the total dosage of theactive agent(s) to be delivered by the pharmaceutical formulation. Forexample, the immediate-release component may provide about 20-60%, orabout 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% of the total dosage ofthe active agent(s) to be delivered by the pharmaceutical formulation.The extended-release component provides about 40%, 45%, 50%, 55%, 60%,65%, 70%, 75% or 80% of the total dosage of the active agent(s) to bedelivered by the formulation. In some embodiments, the immediate-releasecomponent and the extended-release component contain the same activeingredient. In other embodiments, the immediate-release component andthe extended-release component contain different active ingredients(e.g., an analgesic in one component and an antimuscarinic agent inanother component). In some embodiments, the immediate-release componentand the extended-release component each contains an analgesic selectedfrom the group consisting of aspirin, ibuprofen, naproxen sodium,indomethacin, nabumetone, and acetaminophen. In other embodiments, theimmediate-release component and/or the extended-release componentfurther comprises one or more additional active agents selected from thegroups consisting of an antimuscarinic agent, an antidiuretic, aspasmolytic, an inhibitor of phosphodiesterase type (PDE 5 inhibitor)and zolpidem.

In some embodiments, the pharmaceutical composition comprises aplurality of active ingredients selected from the group consisting ofanalgesics, antimuscarinic agents, antidiuretics, spasmolytics and PDE 5inhibitors. Examples of antimuscarinic agents include, but are notlimited to, oxybutynin, solifenacin, darifenacin, fesoterodine,tolterodine, trospium, atropine, and tricyclic antidepressants. Examplesof antidiuretics include, but are not limited to, antidiuretic hormone(ADH), angiotensin II, aldosterone, vasopressin, vasopressin analogs(e.g., desmopressin argipressin, lypressin, felypressin, ornipressin,terlipressin); vasopressin receptor agonists, atrial natriuretic peptide(ANP) and C-type natriuretic peptide (CNP) receptor (i.e., NPR1, NPR2,and NPR3) antagonists (e.g., HS-142-1, isatin, [Asu7,23′]b-ANP-(7-28)],anantin, a cyclic peptide from Streptomyces coerulescens, and 3G12monoclonal antibody); somatostatin type 2 receptor antagonists (e.g.,somatostatin), pharmaceutically-acceptable derivatives, and analogs,salts, hydrates, and solvates thereof. Examples of spasmolytics include,but are not limited to, carisoprodol, benzodiazepines, baclofen,cyclobenzaprine, metaxalone, methocarbamol, clonidine, clonidine analog,and dantrolene. Examples of PDE 5 inhibitors include, but are notlimited to, tadalafil, sildenafil and vardenafil.

In some embodiments, the pharmaceutical composition comprises one ormore analgesics. In other embodiments, the pharmaceutical compositioncomprises (1) one or more analgesics, and (2) one or more other activeingredients selected from the group consisting of antimuscarinic agents,antidiuretics, spasmolytics and PDE 5 inhibitors. In another embodiment,the pharmaceutical composition comprises (1) one or more analgesics and(2) one or more antimuscarinic agents. In another embodiment, thepharmaceutical composition comprises (1) one or more analgesics and (2)one or more antidiuretics. In another embodiment, the pharmaceuticalcomposition comprises (1) one or more analgesics and (2) one or morespasmolytics. In another embodiment, the pharmaceutical compositioncomprises (1) one or more analgesics and (2) one or more PDE 5inhibitors. In another embodiment, the pharmaceutical compositioncomprises (1) one or two analgesics, (2) one or two antimuscarinicagents, and (3) one or two antidiuretics. In another embodiment, thepharmaceutical composition comprises (1) one or two analgesics, (2) oneor two antimuscarinic agents, and (3) one or two spasmolytics. Inanother embodiment, the pharmaceutical composition comprises (1) one ortwo analgesics, (2) one or two antimuscarinic agents, and (3) one or twoPDE 5 inhibitors. In another embodiment, the pharmaceutical compositioncomprises (1) one or more analgesics, (2) one or more antidiuretics, and(3) one or more spasmolytics. In another embodiment, the pharmaceuticalcomposition comprises (1) one or more analgesics, (2) one or moreantidiuretics, and (3) one or more PDE 5 inhibitors. In anotherembodiment, the pharmaceutical composition comprises (1) one or moreanalgesics, (2) one or more spasmolytics, and (3) one or more PDE 5inhibitors.

In one embodiment, the plurality of active ingredients are formulatedfor immediate-release. In other embodiment, the plurality of activeingredients are formulated for extended-release. In other embodiment,the plurality of active ingredients are formulated for bothimmediate-release and extended-release (e.g., a first portion of eachactive ingredient is formulated for immediate-release and a secondportion of each active ingredient is formulated for extended-release).In yet other embodiment, some of the plurality of active ingredients areformulated for immediate-release and some of the plurality of activeingredients are formulated for extended-release (e.g., activeingredients A, B, C are formulated for immediate-release and activeingredients C and D are formulated for extended-release). In some otherembodiments, the immediate-release component and/or the extended-releasecomponent is further coated with a delayed-release coating, such as anenteric coating.

In certain embodiments, the pharmaceutical composition comprises animmediate-release component and an extended-release component. Theimmediate-release component may comprise one or more active ingredientsselected from the group consisting of analgesics, antimuscarinic agents,antidiuretics, spasmolytics and PDE 5 inhibitors. The extended-releasecomponent may comprise one or more active ingredients selected from thegroup consisting of analgesics, antimuscarinic agents, antidiuretics,spasmolytics, PDE 5 inhibitors and zolpidem. In some embodiments, theimmediate-release component and the extended-release component haveexactly the same active ingredients. In other embodiments, theimmediate-release component and the extended-release component havedifferent active ingredients. In yet other embodiments, theimmediate-release component and the extended-release component have oneor more common active ingredients. In some other embodiments, theimmediate-release component and/or the extended-release component isfurther coated with a delayed-release coating, such as an entericcoating.

In one embodiment, the pharmaceutical composition comprises two or moreactive ingredients (e.g., two or more analgesic agents or a mixture ofone or more analgesic agent and one or more antimuscarinic agents orantidiuretics or spasmolytics or PDE 5 inhibitors), formulated forimmediate-release at about the same time. In another embodiment, thepharmaceutical composition comprises two or more active ingredients,formulated for extended-release at about the same time. In anotherembodiment, the pharmaceutical composition comprises two or more activeingredients formulated as two extended-release components, eachproviding a different extended-release profile. For example, a firstextended-release component releases a first active ingredient at a firstrelease rate and a second extended-release component releases a secondactive ingredient at a second release rate. In another embodiment, thepharmaceutical composition comprises two or more active ingredients,both formulated for delayed release.

In another embodiment, the pharmaceutical composition comprises two ormore active ingredients formulated for delayed release. In anotherembodiment, the pharmaceutical composition comprises two or more activeingredients formulated as two delayed-release components, each providinga different delayed-release profile. For example, a firstdelayed-release component releases a first active ingredient at a firsttime point, and a second delayed-release component releases a secondactive ingredient at a second time point.

In other embodiments, the pharmaceutical composition comprises twoactive ingredients (e.g., two analgesic agents, or a mixture of oneanalgesic agent and one antimuscarinic agent or antidiuretic orspasmolytic or a PDE 5 inhibitor or zolpidem) formulated forimmediate-release and (2) two active ingredients (e.g., two analgesicagents, or a mixture of one analgesic agent and one antimuscarinic agentor antidiuretic or spasmolytic or PDE 5 inhibitors) formulated forextended-release. In other embodiments, the pharmaceutical compositioncomprises three active ingredients formulated for immediate-release and(2) three active ingredients formulated for extended-release. In otherembodiments, the pharmaceutical composition comprises four activeingredients formulated for immediate-release and (2) four activeingredients formulated for extended-release. In these embodiments, theactive ingredient(s) in the immediate-release component can be the sameas, or different from, the active ingredient(s) in the extended-releasecomponent. In some other embodiments, the immediate-release componentand/or the extended-release component is further coated with adelayed-release coating, such as an enteric coating.

In some embodiments, the pharmaceutical composition comprises one ormore analgesic agents and a PDE 5 inhibitor, wherein the one or moreanalgesic agents are formulated for delayed release and wherein the PDE5 inhibitor is formulated for immediate release. In other embodiments,the pharmaceutical composition further comprises an additional agentselected from the group consisting of antimuscarinic agents,antidiuretics, spasmolytics, PDE 5 inhibitors and zolpidem, wherein theadditional agent is formulated for immediate-release or delayed-release.In some embodiments, the delayed-release formulation delays the releaseof the active ingredient (e.g., the analgesic agent, antimuscarinicagent, antidiuretic, spasmolytic, zolpidem and/or PDE 5 inhibitor) for aperiod of 1, 2, 3, 4 or 5 hours.

An immediate-release composition may comprise 100% of the total dosageof a given active agent administered in a single unit dose.Alternatively, an immediate-release component may be included as acomponent in a combined release profile formulation that may provideabout 1% to about 60% of the total dosage of the active agent(s) to bedelivered by the pharmaceutical formulation. For example, theimmediate-release component may provide about 5%-60%, about 10% to about60%, about 10% to about 50%, about 10% to about 40%, about 10% to about30%, about 10% to about 20%, about 20% to about 60%, about 20% to about50%, about 20% to about 30%, about 30% to about 60%, about 30% to about50%, about 40% to about 60%, about 40% to about 50%, about 45% to about60% or about 45% to about 50% of the total dosage of the active agent(s)to be delivered by the formulation. In alternate embodiments, theimmediate-release component provides about 2, 4, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55 or 60% of the total dosage of the active agent(s) tobe delivered by the formulation.

In some embodiments, the immediate-release or delayed-releaseformulation comprises an active core comprised of one or more inertparticles, each in the form of a bead, pellet, pill, granular particle,microcapsule, microsphere, microgranule, nanocapsule, or nanospherecoated on its surfaces with drugs in the form of e.g., a drug-containingfilm-forming composition using, for example, fluid bed techniques orother methodologies known to those of skill in the art. The inertparticle can be of various sizes, so long as it is large enough toremain poorly dissolved. Alternatively, the active core may be preparedby granulating and milling and/or by extrusion and spheronization of apolymer composition containing the drug substance.

The amount of drug in the core will depend on the dose that is requiredand typically varies from about 5 to 90 weight %. Generally, thepolymeric coating on the active core will be from about 1 to 50% basedon the weight of the coated particle, depending on the lag time and typeof release profile required and/or the polymers and coating solventschosen. Those skilled in the art will be able to select an appropriateamount of drug for coating onto or incorporating into the core toachieve the desired dosage. In one embodiment, the inactive core may bea sugar sphere or a buffer crystal or an encapsulated buffer crystalsuch as calcium carbonate, sodium bicarbonate, fumaric acid, tartaricacid, etc. which alters the microenvironment of the drug to facilitateits release.

In some embodiments, the delayed-release formulation is formed bycoating a water soluble/dispersible drug-containing particle, such as abead, with a mixture of a water insoluble polymer and an entericpolymer, wherein the water insoluble polymer and the enteric polymer maybe present at a weight ratio of 4:1 to 1:1, and the total weight of thecoatings is 10 to 60 weight % based on the total weight of the coatedbeads. The drug layered beads may optionally include an innerdissolution rate controlling membrane of ethylcellulose. The compositionof the outer layer, as well as the individual weights of the inner andouter layers of the polymeric membrane are optimized for achievingdesired circadian rhythm release profiles for a given active, which arepredicted based on in vitro/in vivo correlations.

In other embodiments the formulations comprise a mixture ofimmediate-release drug-containing particles without a dissolution ratecontrolling polymer membrane and delayed release beads exhibiting, forexample, a lag time of 2-4 hours following oral administration, thusproviding a two-pulse release profile. In yet other embodiments theformulations comprise a mixture of two types of delayed-release beads: afirst type that exhibits a lag time of 1-3 hours and a second type thatexhibits a lag time of 4-6 hours. In yet other embodiments theformulations comprise a mixture of two types of release beads: a firsttype that exhibits immediate-release and a second type that exhibits alag time of 1-4 hours followed with extended-release.

In other embodiments, the formulations are designed with a releaseprofile such that a fraction of the medicine (e.g., 20-60%) is releasedimmediately or within two hours of administration, and the rest isreleased over an extended period of time. The pharmaceutical compositionmay be administered daily or administered on an as needed basis. Incertain embodiments, the pharmaceutical composition is administered tothe subject prior to bedtime. In some embodiments, the pharmaceuticalcomposition is administered immediately before bedtime. In someembodiments, the pharmaceutical composition is administered within abouttwo hours before bedtime, preferably within about one hour beforebedtime. In another embodiment, the pharmaceutical composition isadministered about two hours before bedtime. In a further embodiment,the pharmaceutical composition is administered at least two hours beforebedtime. In another embodiment, the pharmaceutical composition isadministered about one hour before bedtime. In a further embodiment, thepharmaceutical composition is administered at least one hour beforebedtime. In still another embodiment, the pharmaceutical composition isadministered immediately before bedtime. Preferably, the pharmaceuticalcomposition is administered orally.

The appropriate dosage (“therapeutically effective amount”) of theactive agent(s) in the immediate-release component, the extended-releasecomponent, the delayed-release component or delayed-extended-releasecomponent will depend, for example, on the severity and course of thecondition, the mode of administration, the bioavailability of theparticular agent(s), the age and weight of the patient, the patient'sclinical history and response to the active agent(s), discretion of thephysician, etc.

As a general proposition, the therapeutically effective amount of theanalgesic agent(s) in the immediate-release component, thedelayed-release component, the extended-release component or thedelayed-extended-release component is administered in the range of about10 μg/kg body weight/day to about 100 mg/kg body weight/day whether byone or more administrations. In some embodiments, the range of eachactive agent administered daily in a single dose or in multiple does isfrom about 10 μg/kg body weight/day to about 100 mg/kg body weight/day,10 μg/kg body weight/day to about 30 mg/kg body weight/day, 10 μg/kgbody weight/day to about 10 mg/kg body weight/day, 10 μg/kg bodyweight/day to about 3 mg/kg body weight/day, 10 μg/kg body weight/day toabout 1 mg/kg body weight/day, 10 μg/kg body weight/day to about 300μg/kg body weight/day, 10 μg/kg body weight/day to about 100 μg/kg bodyweight/day, 10 μg/kg body weight/day to about 30 μg/kg body weight/day,30 μg/kg body weight/day to about 100 mg/kg body weight/day, 30 μg/kgbody weight/day to about 30 mg/kg body weight/day, 30 μg/kg bodyweight/day to about 10 mg/kg body weight/day, 30 μg/kg body weight/dayto about 3 mg/kg body weight/day, 30 μg/kg body weight/day to about 1mg/kg body weight/day, 30 μg/kg body weight/day to about 300 μg/kg bodyweight/day, 30 μg/kg body weight/day to about 100 μg/kg body weight/day,100 μg/kg body weight/day to about 100 mg/kg body weight/day, 100 μg/kgbody weight/day to about 30 mg/kg body weight/day, 100 μg/kg bodyweight/day to about 10 mg/kg body weight/day, 100 μg/kg body weight/dayto about 3 mg/kg body weight/day, 100 μg/kg body weight/day to about 1mg/kg body weight/day, 100 μg/kg body weight/day to about 300 μg/kg bodyweight/day, 300 μg/kg body weight/day to about 100 mg/kg bodyweight/day, 300 μg/kg body weight/day to about 30 mg/kg body weight/day,300 μg/kg body weight/day to about 10 mg/kg body weight/day, 300 μg/kgbody weight/day to about 3 mg/kg body weight/day, 300 μg/kg bodyweight/day to about 1 mg/kg body weight/day, 1 mg/kg body weight/day toabout 100 mg/kg body weight/day, 1 mg/kg body weight/day to about 30mg/kg body weight/day, 1 mg/kg body weight/day to about 10 mg/kg bodyweight/day, 1 mg/kg body weight/day to about 3 mg/kg body weight/day, 3mg/kg body weight/day to about 100 mg/kg body weight/day, 3 mg/kg bodyweight/day to about 30 mg/kg body weight/day, 3 mg/kg body weight/day toabout 10 mg/kg body weight/day, 10 mg/kg body weight/day to about 100mg/kg body weight/day, 10 mg/kg body weight/day to about 30 mg/kg bodyweight/day or 30 mg/kg body weight/day to about 100 mg/kg bodyweight/day.

The analgesic agent(s) described herein may be included in animmediate-release component or an extended-release component, adelayed-release component, a delayed-extended-release component orcombinations thereof for daily oral administration at a single dose orcombined dose range of 1 mg to 2000 mg, 1 mg to 1000 mg, 1 mg to 300 mg,1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 10 mg, 1 mg to 3 mg, 3 mg to 2000mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mgto 10 mg, 10 mg to 2000 mg, 10 mg to 1000 mg, 10 mg to 300 mg, 10 mg to100 mg, 10 mg to 30 mg, 30 mg to 2000 mg, 30 mg to 1000 mg, 30 mg to 300mg, 30 mg to 100 mg, 100 mg to 2000 mg, 100 mg to 1000 mg, 100 mg to 300mg, 300 mg to 2000 mg, 300 mg to 1000 mg or 1000 mg to 2000 mg. Asexpected, the dosage will be dependent on the condition, size, age, andcondition of the patient.

In some embodiments, the pharmaceutical composition comprises a singleanalgesic agent. In one embodiment, the single analgesic agent isaspirin. In another embodiment, the single analgesic agent is ibuprofen.In another embodiment, the single analgesic agent is naproxen ornaproxen sodium. In another embodiment, the single analgesic agent isindomethacin. In another embodiment, the single analgesic agent isnabumetone. In another embodiment, the single analgesic agent isacetaminophen.

In other embodiments, the pharmaceutical composition comprises a pair ofanalgesic agents. Examples of such paired analgesic agents include, butare not limited to, acetylsalicylic acid and ibuprofen, acetylsalicylicacid and naproxen sodium, acetylsalicylic acid and nabumetone,acetylsalicylic acid and acetaminophen, acetylsalicylic acid andindomethancin, ibuprofen and naproxen sodium, ibuprofen and nabumetone,ibuprofen and acetaminophen, ibuprofen and indomethancin, naproxen,naproxen sodium and nabumetone, naproxen sodium and acetaminophen,naproxen sodium and indomethancin, nabumetone and acetaminophen,nabumetone and indomethancin, and acetaminophen and indomethancin. Thepaired analgesic agents are mixed at a weight ratio in the range of0.1:1 to 10:1, 0.2:1 to 5:1 or 0.3:1 to 3:1. In one embodiment, thepaired analgesic agents are mixed at a weight ratio of 1:1.

In some other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more antimuscarinic agents.Examples of the antimuscarinic agents include, but are not limited to,oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine,trospium, atropine, and tricyclic antidepressants. The daily dose ofantimuscarinic agent is in the range of 1 μg to 300 mg, 1 μg to 100 mg,1 μg to 30 mg; 1 μg to 10 mg, 1 μg to 3 mg, 1 μg to 1 mg, 1 μg to 300μg, 1 μg to 100 μg, 1 μg to 30 μg, 1 μg to 10 μg, 1 μg to 3 μg, 3 μg to100 mg, 3 μg to 100 mg, 3 μg to 30 mg; 3 μg to 10 mg, 3 μg to 3 mg, 3 μgto 1 mg, 3 μg to 300 μg, 3 μg to 100 μg, 3 μg to 30 μg, 3 μg to 10 μg,10 μg to 300 mg, 10 μg to 100 mg, 10 μg to 30 mg; 10 μg to 10 mg, 10 μgto 3 mg, 10 μg to 1 mg, 10 μg to 300 μg, 10 μg to 100 μg, 10 μg to 30μg, 30 μg to 300 mg, 30 μg to 100 mg, 30 μg to 30 mg; 30 μg to 10 mg, 30μg to 3 mg, 30 μg to 1 mg, 30 μg to 300 μg, 30 μg to 100 μg, 100 μg to300 mg, 100 μg to 100 mg, 100 μg to 30 mg; 100 μg to 10 mg, 100 μg to 3mg, 100 μg to 1 mg, 100 μg to 300 μg, 300 μg to 300 mg, 300 μg to 100mg, 300 μg to 30 mg; 300 μg to 10 mg, 300 μg to 3 mg, 300 μg to 1 mg, 1mg to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 3 mg, 3 mg to 300mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 300 mg, 10 mgto 100 mg, 10 mg to 30 mg, 30 mg to 300 mg, 30 mg to 100 mg or 100 mg to300 mg.

In some other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more antidiuretics. Examples of theantidiuretics include, but are not limited to, antidiuretic hormone(ADH), angiotensin II, aldosterone, vasopressin, vasopressin analogs(e.g., desmopressin argipressin, lypressin, felypressin, ornipressin,and terlipressin), vasopressin receptor agonists, atrial natriureticpeptide (ANP) and C-type natriuretic peptide (CNP) receptor (i.e., NPR1,NPR2, and NPR3) antagonists (e.g., HS-142-1, isatin,[Asu7,23′]b-ANP-(7-28)], anantin, a cyclic peptide from Streptomycescoerulescens, and 3G12 monoclonal antibody), somatostatin type 2receptor antagonists (e.g., somatostatin), pharmaceutically-acceptablederivatives, and analogs, salts, hydrates, and solvates thereof. In someembodiments, the one or more antidiuretics comprise desmopressin. Inother embodiments, the one or more antidiuretics is desmopressin. Thedaily dose of antidiuretic is in the range of 1 μg to 300 mg, 1 μg to100 mg, 1 μg to 30 mg; 1 μg to 10 mg, 1 μg to 3 mg, 1 μg to 1 mg, 1 μgto 300 μg, 1 μg to 100 μg, 1 μg to 30 μg, 1 μg to 10 μg, 1 μg to 3 μg, 3μg to 100 mg, 3 μg to 100 mg, 3 μg to 30 mg; 3 μg to 10 mg, 3 μg to 3mg, 3 μg to 1 mg, 3 μg to 300 μg, 3 μg to 100 μg, 3 μg to 30 μg, 3 μg to10 μg, 10 μg to 300 mg, 10 μg to 100 mg, 10 μg to 30 mg; 10 μg to 10 mg,10 μg to 3 mg, 10 μg to 1 mg, 10 μg to 300 μg, 10 μg to 100 μg, 10 μg to30 μg, 30 μg to 300 mg, 30 μg to 100 mg, 30 μg to 30 mg; 30 μg to 10 mg,30 μg to 3 mg, 30 μg to 1 mg, 30 μg to 300 μg, 30 μg to 100 μg, 100 μgto 300 mg, 100 μg to 100 mg, 100 μg to 30 mg; 100 μg to 10 mg, 100 μg to3 mg, 100 μg to 1 mg, 100 μg to 300 μg, 300 μg to 300 mg, 300 μg to 100mg, 300 μg to 30 mg; 300 μg to 10 mg, 300 μg to 3 mg, 300 μg to 1 mg, 1mg to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 3 mg, 3 mg to 300mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 300 mg, 10 mgto 100 mg, 10 mg to 30 mg, 30 mg to 300 mg, 30 mg to 100 mg or 100 mg to300 mg.

In other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more spasmolytics. Examples ofspasmolytics include, but are not limited to, carisoprodol,benzodiazepines, baclofen, cyclobenzaprine, metaxalone, methocarbamol,clonidine, clonidine analog, and dantrolene. In some embodiments, thespasmolytics is used at a daily dose of 0.1 mg to 1000 mg, 0.1 mg to 300mg, 0.1 mg to 100 mg, 0.1 mg to 30 mg, 0.1 mg to 10 mg, 0.1 mg to 3 mg,0.1 mg to 1 mg, 0.1 mg to 0.3 mg, 0.3 mg to 1000 mg, 0.3 mg to 300 mg,0.3 mg to 100 mg, 0.3 mg to 30 mg, 0.3 mg to 10 mg, 0.3 mg to 3 mg, 0.3mg to 1 mg, 1 mg to 1000 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to 30mg, 1 mg to 10 mg, 1 mg to 3 mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mgto 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 1000 mg, 10 mg to 300mg, 10 mg to 100 mg, 10 mg to 30 mg, 30 mg to 1000 mg, 30 mg to 300 mg,30 mg to 100 mg, 100 mg to 1000 mg, 100 mg to 300 mg, or 300 mg to 1000mg.

In other embodiments, the pharmaceutical composition of the presentapplication further comprises one or more PDE 5 inhibitors. Examples ofPDE 5 inhibitors include, but are not limited to, tadalafil, sildenafiland vardenafil. In some embodiments, the one or more PDE 5 inhibitorscomprise tadalafil. In other embodiments, the one or more PDE 5inhibitors is tadalafil. In some embodiments, the PDE 5 inhibitor isused at a daily dose of 0.1 mg to 1000 mg, 0.1 mg to 300 mg, 0.1 mg to100 mg, 0.1 mg to 30 mg, 0.1 mg to 10 mg, 0.1 mg to 3 mg, 0.1 mg to 1mg, 0.1 mg to 0.3 mg, 0.3 mg to 1000 mg, 0.3 mg to 300 mg, 0.3 mg to 100mg, 0.3 mg to 30 mg, 0.3 mg to 10 mg, 0.3 mg to 3 mg, 0.3 mg to 1 mg, 1mg to 1000 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 10mg, 1 mg to 3 mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mgto 30 mg, 3 mg to 10 mg, 10 mg to 1000 mg, 10 mg to 300 mg, 10 mg to 100mg, 10 mg to 30 mg, 30 mg to 1000 mg, 30 mg to 300 mg, 30 mg to 100 mg,100 mg to 1000 mg, 100 mg to 300 mg, or 300 mg to 1000 mg.

In some other embodiments, the pharmaceutical composition of the presentapplication further comprises zolpidem. The daily dose of zolpidem is inthe range of 100 μg to 100 mg, 100 μg to 30 mg, 100 μg to 10 mg, 100 μgto 3 mg, 100 μg to 1 mg, 100 μg to 300 μg, 300 μg to 100 mg, 300 μg to30 mg, 300 μg to 10 mg, 300 μg to 3 mg, 300 μg to 1 mg, 1 mg to 100 mg,1 mg to 30 mg, 1 mg to 10 mg, 1 mg to 3 mg, 10 mg to 100 mg, 10 mg to 30mg, or 30 mg to 100 mg.

The antimuscarinic agents, antidiuretics, spasmolytics, zolpidem and/orPDE 5 inhibitors may be formulated, alone or together with other activeingredient(s) in the pharmaceutical composition, for immediate-release,extended-release, delayed release, delayed-extended-release orcombinations thereof.

In certain embodiments, the pharmaceutical composition is formulated forextended release and comprises (1) an analgesic agent selected from thegroup consisting of cetylsalicylic acid, ibuprofen, naproxen, naproxensodium, nabumetone, acetaminophen, and indomethancin and (2) a PDE 5inhibitor, such as tadalafil.

The pharmaceutical composition may be formulated into a tablet, capsule,dragee, powder, granulate, liquid, gel or emulsion form. Said liquid,gel or emulsion may be ingested by the subject in naked form orcontained within a capsule.

In some embodiments, the pharmaceutical composition comprises a singleanalgesic agent and a single PDE 5 inhibitor. In one embodiment, thesingle analgesic agent is aspirin. In another embodiment, the singleanalgesic agent is ibuprofen. In another embodiment, the singleanalgesic agent is naproxen or naproxen sodium. In another embodiment,the single analgesic agent is indomethacin. In another embodiment, thesingle analgesic agent is nabumetone. In another embodiment, the singleanalgesic agent is acetaminophen. In another embodiment, the single PDE5 inhibitor is tadalafil. The analgesic agent and PDE 5 inhibitor may begiven at doses in the ranges described above.

In some embodiments, the pharmaceutical composition comprises one ormore analgesic agents, individually or in combination, in an amountbetween 10-1000 mg, 10-800 mg, 10-600 mg, 10-500 mg, 10-400 mg, 10-300mg, 10-250 mg, 10-200 mg, 10-150 mg, 10-100 mg 30-1000 mg, 30-800 mg,30-600 mg, 30-500 mg, 30-400 mg, 30-300 mg, 30-250 mg, 30-200 mg, 30-150mg, 30-100 mg, 100-1000 mg, 100-800 mg, 100-600 mg, 100-400 mg, 100-250mg, 300-1000 mg, 300-800 mg, 300-600 mg, 300-400 mg, 400-1000 mg,400-800 mg, 400-600 mg, 600-1000 mg, 600-800 mg or 800-1000 mg, whereinthe composition is formulated for extended release with a releaseprofile in which the one or more analgesic agents are releasedcontinuously over a period of 2-12 hours or 5-8 hours.

In some embodiments, the composition is formulated for extended releasewith a release profile in which at least 90% of the one or moreanalgesic agents are released continuously over a period of 2-12 hoursor 5-8 hours.

In some embodiments, the composition is formulated for extended releasewith a release profile in which the one or more analgesic agents arereleased continuously over a period of 5, 6, 7, 8, 10 or 12 hours. Insome embodiments, the pharmaceutical composition further comprises anantimuscarinic agent, an antidiuretic, a spasmolytic, zolpidem or a PDE5 inhibitor.

In other embodiments, the composition is formulated for extended releasewith a release profile in which the analgesic agent is released at asteady rate over a period of 2-12 hours or 5-8 hours. In otherembodiments, the composition is formulated for extended release with arelease profile in which the analgesic agent is released at a steadyrate over a period of 5, 6, 7, 8, 10 or 12 hours. As used herein, “asteady rate over a period of time” is defined as a release profile inwhich the release rate at any point during a given period of time iswithin 30%-300% of the average release rate over that given period oftime. For example, if 80 mg of aspirin is released at a steady rate overa period of 8 hours, the average release rate is 10 mg/hr during thisperiod of time and the actual release rate at any time during thisperiod is within the range of 3 mg/hr to 30 mg/hr (i.e., within 30%-300%of the average release rate of 10 mg/hr during the 8 hour period). Insome embodiments, the pharmaceutical composition further comprises anantimuscarinic agent, an antidiuretic a spasmolytic, zolpidem or a PDE 5inhibitor.

In some embodiments, the analgesic agent is selected from the groupconsisting of aspirin, ibuprofen, naproxen sodium, naproxen,indomethacin, nabumetone and acetaminophen. In one embodiment, theanalgesic agent is acetaminophen. The pharmaceutical composition isformulated to provide a steady release of small amount of the analgesicagent to maintain an effective drug concentration in the blood such thatthe overall amount of the drug in a single dosage is reduced compared tothe immediate release formulation.

In some other embodiments, the pharmaceutical composition comprises oneor more analgesic agent(s), individually or in combination, in an amountbetween 10-1000 mg, 10-800 mg, 10-600 mg, 10-500 mg, 10-400 mg, 10-300mg, 10-250 mg, 10-200 mg, 10-150 mg, 10-100 mg 30-1000 mg, 30-800 mg,30-600 mg, 30-500 mg, 30-400 mg, 30-300 mg, 30-250 mg, 30-200 mg, 30-150mg, 30-100 mg, 100-1000 mg, 100-800 mg, 100-600 mg, 100-400 mg, 100-250mg, 300-1000 mg, 300-800 mg, 300-600 mg, 300-400 mg, 400-1000 mg,400-800 mg, 400-600 mg, 600-1000 mg, 600-800 mg or 800-1000 mg, whereinthe analgesic agent(s) are formulated for extended release,characterized by a two-phase release profile in which 20-60% of theanalgesic agent(s) are released within 2 hours of administration and theremainder are released continuously, or at a steady rate, over a periodof 2-12 hours or 5-8 hours. In yet another embodiment, the analgesicagent(s) is formulated for extended release with a two-phase releaseprofile in which 20, 30, 40, 50 or 60% of the analgesic agent(s) arereleased within 2 hours of administration and the remainder are releasedcontinuously, or at a steady rate, over a period of 2-12 hours or 5-8hours. In one embodiment, the analgesic agent(s) are selected from thegroup consisting of aspirin, ibuprofen, naproxen sodium, naproxen,indomethacin, nabumetone, and acetaminophen. In one embodiment, theanalgesic agent is acetaminophen. In another embodiment, the analgesicagent is acetaminophen. In some embodiments, the pharmaceuticalcomposition further comprises an antimuscarinic agent, an antidiuretic,a spasmolytic, zolpidem and/or a PDE 5 inhibitor. In some embodiments,the antimuscarinic agent, antidiuretic, spasmolytic, zolpidem and/or PDE5 inhibitor is/are formulated for immediate-release.

Another aspect of the present application relates to a method forreducing frequency of urination by administering to a subject in needthereof, two or more analgesic agents alternatively to prevent thedevelopment of drug resistance. In one embodiment, the method comprisesadministering a first analgesic agent for a first period of time andthen administering a second analgesic agent for a second period of time.In another embodiment, the method further comprises administering athird analgesic agent for a third period of time. The first, second, andthird analgesic agents are different from each other and at least one ofwhich is formulated for extended-release or delayed, extended-release.In one embodiment, the first analgesic agent is acetaminophen, thesecond analgesic agent is ibuprofen, and the third analgesic agent isnaproxen sodium. The length of each period may vary depending on thesubject's response to each analgesic agent. In some embodiments, eachperiod lasts from 3 days to three weeks. In another embodiment, thefirst, second, and third analgesic are all formulated forextended-release or delayed, extended-release.

Another aspect of the present application relates to a method forreducing frequency of urination by administering to a subject in needthereof, two or more analgesic agents alternatively to prevent thedevelopment of drug resistance. In one embodiment, the method comprisesadministering a first analgesic agent for a first period of time andthen administering a second analgesic agent for a second period of time.In another embodiment, the method further comprises administering athird analgesic agent for a third period of time. The first, second andthird analgesic agents are different from each other and at least one ofwhich is formulated for extended-release or delayed, extended-release.In one embodiment, the first analgesic agent is acetaminophen, thesecond analgesic agent is ibuprofen and the third analgesic agent isnaproxen sodium. The length of each period may vary depending on thesubject's response to each analgesic agent. In some embodiments, eachperiod lasts from 3 days to three weeks. In another embodiment, thefirst, second and third analgesic are all formulated forextended-release or delayed, extended-release.

Another aspect of the present application relates to a method fortreating nocturia by administering to a person in need thereof adiuretic, followed with the pharmaceutical composition of the presentapplication. The diuretic is dosed and formulated to have a diureticeffect within 6 hours of administration and is administered at least 8or 7 hours prior to bedtime. The pharmaceutical composition of thepresent application is formulated for extended-release or delayed,extended-release, and is administered within 2 hours prior to bedtime.

Examples of diuretics include, but are not limited to, acidifying salts,such as CaCl₂ and NH₄Cl; arginine vasopressin receptor 2 antagonists,such as amphotericin B and lithium citrate; aquaretics, such asGoldenrod and Junipe; Na—H exchanger antagonists, such as dopamine;carbonic anhydrase inhibitors, such as acetazolamide and dorzolamide;loop diuretics, such as bumetanide, ethacrynic acid, furosemide andtorsemide; osmotic diuretics, such as glucose and mannitol;potassium-sparing diuretics, such as amiloride, spironolactone,triamterene, potassium canrenoate; thiazides, such asbendroflumethiazide and hydrochlorothiazide; and xanthines, such ascaffeine, theophylline and theobromine.

Another aspect of the present application relates to a method forreducing the frequency of urination, comprising administering to asubject in need thereof an effective amount of botulinum toxin, whereinthe botulinum toxin is administered by injection into a bladder muscle;and orally administering to the subject the pharmaceutical compositionof the present application. In some embodiments, the injecting stepcomprises injection of 10-200 units of botulinum toxin at 5-20 sites inbladder muscle with an injection dose of 2-10 units per site. In oneembodiment, the injecting step comprises injection of botulinum toxin at5 sites in bladder muscle with an injection dose of 2-10 units per site.In another embodiment, the injecting step comprises injection ofbotulinum toxin at 10 sites in bladder muscle at an injection dose of2-10 units per site. In another embodiment, the injecting step comprisesinjection of botulinum toxin at 15 sites in bladder muscle at aninjection dose of 2-10 units per site. In yet another embodiment, theinjecting step comprises injection of botulinum toxin at 20 sites inbladder muscle at an injection dose of 2-10 units per site. In someembodiments, the injecting step is repeated every 3, 4, 6, 8, 10 or 12months and the oral administration step is repeated daily.

In some embodiments, the pharmaceutical composition of the presentapplication comprises an active ingredient comprising one or moreanalgesic agents in an amount of 50-400 mg per agent, wherein the one ormore analgesic agents are selected from the group consisting of aspirin,ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, andacetaminophen, wherein the pharmaceutical composition is formulated forextended-release. In other embodiments, the botulinum toxin isadministered every 3 or 4 months and the pharmaceutical composition ofthe present application is administered daily. The method can be usedfor the treatment of nocturia or overactive bladder.

Another aspect of the present application relates to a method forreducing frequency of urination in a subject. The method comprisesadministering to a subject in need thereof an effective amount of one ormore analgesic agents and an effective amount of tadalafil.

In one embodiment, the one or more analgesic agents are formulated forextended release and the tadalafil is formulated for immediate release.

In another embodiment, the one or more analgesic agents are formulatedfor delayed release and the tadalafil is formulated for immediaterelease.

Another aspect of the present application relates to a method forreducing frequency of urination in a subject. The method comprisesadministering to a subject in need thereof a pharmaceutical compositioncomprising an active ingredient comprising one or more analgesic agentsin an amount of 1-2000 mg per agent; and a PDE 5 inhibitor, wherein theone or more analgesic agents are selected from the group consisting ofaspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone,and acetaminophen.

In one embodiment, the pharmaceutical composition is coated with anenteric coating.

In another embodiment, the pharmaceutical composition is formulated forextended release, characterized by a two-phase release profile in which20-60% of the active ingredient is released within two hours ofadministration and remainder of said active ingredient is releasedcontinuously over a period of 2-12 hours. In a related embodiment, thepharmaceutical composition is coated with an enteric coating.

In another embodiment, one or more analgesic agents comprisesacetaminophen.

In another embodiment, the active ingredient further comprises anadditional agent selected from the group consisting of antimuscarinicagents, antidiuretics, spasmolytics and zolpidem.

In another embodiment, the PDE 5 inhibitor is tadalafil.

Another aspect of the present application relates to a method forreducing frequency of urination in a subject. The method comprisesadministering to a subject in need thereof a pharmaceutical compositioncomprising: a first active ingredient comprising one or more analgesicagents and tadalafil; and a second active ingredient comprising one ormore agents selected from the group consisting of analgesic agents,antimuscarinic agents, antidiuretics, spasmolytics, PDE 5 inhibitors andzolpidem, wherein the first active ingredient is formulated forimmediate release and wherein the second active ingredient is formulatedfor extended release.

In one embodiment, the pharmaceutical composition is further coated withan enteric coating.

In another embodiment, the first active ingredient comprisesacetaminophen.

In another embodiment, the first active ingredient further comprises anantimuscarinic agent, an antidiuretic, a spasmolytic or zolpidem.

Another aspect of the present application relates to a pharmaceuticalcomposition, comprising one or more analgesic agents, a PDE 5 inhibitorand a pharmaceutically acceptable carrier.

In one embodiment, the one or more analgesic agents are formulated forextended-release the PDE 5 inhibitor is formulated forimmediate-release.

In another embodiment, the one or more analgesic agents are formulatedfor delayed-release and the PDE 5 inhibitor is formulated forimmediate-release.

In another embodiment, the one or more analgesic agents and said PDE 5inhibitor are formulated for extended release over a period of 2-12hours.

In another embodiment, the PDE 5 inhibitor and 20-60% of each of the oneor more analgesic agents are formulated for immediate release, andwherein remainder of each of said one or more analgesic agents isformulated for extended release. In a related embodiment, thepharmaceutical composition is further coated with an enteric coating.

The present invention is further illustrated by the following examplewhich should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application are incorporated herein by reference.

Example 1 Inhibition of the Urge to Urinate

Twenty volunteer subjects, both male and female were enrolled, each ofwhich experienced premature urge or desire to urinate, interfering withtheir ability to sleep for a sufficient period of time to feeladequately rested. Each subject ingested 400-800 mg of ibuprofen as asingle dose prior to bedtime. At least 14 subjects reported that theywere able to rest better because they were not being awakened asfrequently by the urge to urinate.

Several subjects reported that after several weeks of nightly use ofibuprofen, the benefit of less frequent urges to urinate was no longerbeing realized. However, all of these subjects further reported thereturn of the benefit after several days of abstaining from taking thedosages. More recent testing has confirmed similar results can beachieved at much lower dosages without any subsequent diminution ofbenefits.

Example 2 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Macrophage Responses to Inflammatory andNon-Inflammatory Stimuli Experimental Design

This study is designed to determine the dose and in vitro efficacy ofanalgesics and antimuscarinic agents in controlling macrophage responseto inflammatory and non-inflammatory stimuli mediated by COX2 andprostaglandins (PGE, PGH, etc.). It establishes baseline (dose andkinetic) responses to inflammatory and non-inflammatory effectors inbladder cells. Briefly, cultured cells are exposed to analgesic agentsand/or antimuscarinic agents in the absence or presence of variouseffectors.

The effectors include: lipopolysaccharide (LPS), an inflammatory agent,and Cox2 inducer as inflammatory stimuli; carbachol or acetylcholine,stimulators of smooth muscle contraction as non-inflammatory stimuli;botulinum neurotoxin A, a known inhibitor of acetylcholine release, aspositive control; and arachidonic acid (AA), gamma linolenic acid(DGLA), or eicosapentaenoic acid (EPA) as precursors of prostaglandins,which are produced following the sequential oxidation of AA, DGLA, orEPA inside the cell by cyclooxygenases (COX1 and COX2) and terminalprostaglandin synthases.

The analgesic agents include: Salicylates such as aspirin;iso-butyl-propanoic-phenolic acid derivative (ibuprofen) such as Advil,Motrin, Nuprin, and Medipren; naproxen sodium such as Aleve, Anaprox,Antalgin, Feminax Ultra, Flanax, Inza, Midol Extended Relief, Nalgesin,Naposin, Naprelan, Naprogesic, Naprosyn, Naprosyn suspension,EC-Naprosyn, Narocin, Proxen, Synflex and Xenobid; acetic acidderivative such as indomethacin (Indocin); 1-naphthaleneacetic acidderivative such as nabumetone or relafen; N-acetyl-para-aminophenol(APAP) derivative such as acetaminophen or paracetamol (Tylenol); andCelecoxib.

The antimuscarinic agents include: oxybutynin, solifenacin, darifenacin,and atropine.

Macrophages are subjected to short term (1-2 hrs) or long term (24-48hrs) stimulation with:

1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

The cells are then analyzed for the release of PGH₂; PGE; PGE₂;Prostacydin; Thromboxane; IL-1β; IL-6; TNF-α; the COX2 activity; theproduction of cAMP and cGMP; the production of IL-1β, IL-6, TNF-α, andCOX2 mRNA; and surface expression of CD80, CD86, and MEW class IImolecules.

Materials and Methods Macrophage Cells

Murine RAW264.7 or J774 macrophage cells (obtained from ATCC) were usedin this study. Cells were maintained in a culture medium containing RPMI1640 supplemented with 10% fetal bovine serum (FBS), 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin. Cellswere cultured at 37° C. in a 5% CO₂ atmosphere and split (passages) oncea week.

In Vitro Treatment of Macrophage Cells with Analgesics

RAW264.7 macrophage cells were seeded in 96-well plates at a celldensity of 1.5×10⁵ cells per well in 100 μl of the culture medium. Thecells were treated with (1) various concentrations of analgesic(acetaminophen, aspirin, ibuprofen or naproxen), (2) variousconcentrations of lipopolysaccharide (LPS), which is an effector ofinflammatory stimuli to macrophage cells, (3) various concentrations ofcarbachol or acetylcholine, which are effectors of non-inflammatorystimuli, (4) analgesic and LPS or (5) analgesic and carbachol oracetylcholine. Briefly, the analgesics were dissolved in FBS-freeculture medium (i.e RPMI 1640 supplemented with 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin) anddiluted to desired concentrations by serial dilution with the samemedium. For cells treated with analgesic in the absence of LPS, 50 μl ofanalgesic solution and 50 μl of FBS-free culture medium were added toeach well. For cells treated with analgesic in the presence of LPS, 50μl of analgesic solution and 50 μl of LPS (from Salmonella typhimurium)in FBS-free culture medium were added to each well. All conditions weretested in duplicates.

After 24 or 48 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris and stored at −70° C. for analysis of cytokine responses byELISA. The cells were collected and washed by centrifugation (5 min at1,500 rpm at 4° C.) in 500 μl of Phosphate buffer (PBS). Half of thecells were then snap frozen in liquid nitrogen and stored at −70° C. Theremaining cells were stained with fluorescent monoclonal antibodies andanalyzed by flow cytometry.

Flow Cytometry Analysis of Co-Stimulatory Molecule Expression

For flow cytometry analysis, macrophages were diluted in 100 μl of FACSbuffer (phosphate buffered saline (PBS) with 2% bovine serum albumin(BSA) and 0.01% NaN₃) and stained 30 min at 4° C. by addition ofFITC-conjugated anti-CD40, PE-conjugated anti-CD80, PE-conjugatedanti-CD86 antibody, anti MHC class II (I-A^(d)) PE (BD Bioscience).Cells were then washed by centrifugation (5 min at 1,500 rpm at 4° C.)in 300 μl of FACS buffer. After a second wash, cells were re-suspendedin 200 μl of FACS buffer and the percentage of cells expressing a givenmarker (single positive), or a combination of markers (double positive)were analyzed with the aid of an Accuri C6 flow cytometer (BDBiosciences).

Analysis of Cytokine Responses by ELISA

Culture supernatants were subjected to cytokine-specific ELISA todetermine IL-1β, IL-6, and TNF-α responses in cultures of macrophagestreated with analgesic, LPS alone or a combination of LPS and analgesic.The assays were performed on Nunc MaxiSorp Immunoplates (Nunc) coatedovernight with 100 μl of anti-mouse IL-6, TNF-α mAbs (BD Biosciences) orIL-1β mAb (R&D Systems) in 0.1 M sodium bicarbonate buffer (pH 9.5).After two washes with PBS (200 μl per well), 200 μl of PBS 3% BSA wereadded in each well (blocking) and the plates incubated for 2 hours atroom temperature. Plates were washed again two times by addition of 200μl per well, 100 μl of cytokine standards and serial dilutions ofculture supernatants were added in duplicate, and the plates wereincubated overnight at 4° C. Finally, the plates were washed twice andincubated with 100 μl of secondary biotinylated anti-mouse IL-6, TNFαmAbs (BD Biosciences), or IL-1β (R&D Systems) followed byperoxidase-labeled goat anti-biotin mAb (Vector Laboratories). Thecolorimetric reaction was developed by the addition of 2,2′-azino-bis(3)-ethylbenzylthiazoline-6-sulfonic acid (ABTS) substrate and H₂O₂(Sigma) and the absorbance measured at 415 nm with a Victor® Vmultilabel plate reader (PerkinElmer).

Determination of COX2 Activity and the Production of cAMP and cGMP

The COX2 activity in the cultured macrophages is determined bysequential competitive ELISA (R&D Systems). The production of cAMP andcGMP is determined by the cAMP assay and cGMP assay. These assays areperformed routinely in the art.

Results

Table 1 summarizes the experiments performed with Raw 264 macrophagecell line and main findings in terms of the effects of analgesics oncell surface expression of costimulatory molecules CD40 and CD80.Expression of these molecules is stimulated by COX2 and inflammatorysignals and thus, was evaluated to determine functional consequences ofinhibition of COX2.

As shown in Table 2, acetaminophen, aspirin, ibuprofen, and naproxeninhibit basal expression of co-stimulatory molecules CD40 and CD80 bymacrophages at all the tested doses (i.e., 5×10⁵ nM, 5×10⁴ nM, 5×10³ nM,5×10² nM, 50 nM, and 5 nM), except for the highest dose (i.e., 5×10⁶nM), which appears to enhance, rather than inhibit, expression of theco-stimulatory molecules. As shown in FIGS. 1A and 1B, such inhibitoryeffect on CD40 and CD50 expression was observed at analgesic doses aslow as 0.05 nM (i.e., 0.00005 This finding supports the notion that acontrolled release of small doses of analgesic may be preferable toacute delivery of large doses. The experiment also revealed thatacetaminophen, aspirin, ibuprofen, and naproxen have a similarinhibitory effect on LPS induced expression of CD40 and CD80.

TABLE 1 Summary of experiments LPS Salmonella Control typhimuriumAcetaminophen Aspirin Ibuprofen Naproxen TESTS 1 X 2 X Dose responses(0, 5, 50, 1000) ng/mL 3 X Dose responses (0, 5, 50, 500, 5 × 10³, 5 ×10⁴, 5 × 10⁵, 5 × 10⁶) nM 4 X X (5 ng/mL) Dose responses X (50 ng/mL (0,5, 50, 500, 5 × 10³, 5 × 10⁴, X (1000 ng/mL) 5 × 10⁵, 5 × 10⁶) nMANALYSIS a Characterization of activation/stimulatory status: Flowcytometry analysis of CD40, CD80, CD86, and MHC class II b Mediators ofinflammatory responses: ELISA analysis of IL-1β, IL-6, TNF-α

TABLE 2 Summary of main findings Negative Dose analgesic (nM) Effectors% Positive Control LPS 5 ng/ml 5 × 10⁶ 5 × 10⁵ 5 × 10⁴ 5 × 10³ 500 50 5CD40⁺CD80⁺ 20.6 77.8 Acetaminophen CD40⁺CD80⁺ 63 18 12 9.8 8.3 9.5 7.5Aspirin CD40⁺CD80⁺ 44 11 10.3 8.3 8 10.5 7.5 Ibuprofen CD40⁺CD80⁺ ND*6.4 7.7 7.9 6.0 4.9 5.8 Naproxen CD40⁺CD80⁺ 37 9.6 7.7 6.9 7.2 6.8 5.2Analgesic plus LPS Acetaminophen CD40⁺CD80⁺ 95.1 82.7 72.4 68.8 66.866.2 62.1 Aspirin CD40⁺CD80⁺ 84.5 80 78.7 74.7 75.8 70.1 65.7 IbuprofenCD40⁺CD80⁺ ND 67 77.9 72.9 71.1 63.7 60.3 Naproxen CD40⁺CD80⁺ 66.0 74.177.1 71.0 68.8 72 73 *ND: not done (toxicity)

Table 3 summarizes the results of several studies that measured serumlevels of analgesic after oral therapeutic doses in adult humans. Asshown in Table 3, the maximum serum levels of analgesic after an oraltherapeutic dose are in the range of 10⁴ to 10⁵ nM. Therefore, the dosesof analgesic tested in vitro in Table 2 cover the range ofconcentrations achievable in vivo in humans.

TABLE 3 Serum levels of analgesic in human blood after oral therapeuticdoses Maximum serum levels after oral Molecular therapeutic dosesAnalgesic drug weight mg/L nM References Acetaminophen 151.16 11-18  7.2× 10⁴-1.19 × 10⁵ BMC Clinical Pharmacology. 2010, 10:10 (Tylenol)Anaesth Intensive Care. 2011, 39: 242 Aspirin 181.66  30-100 1.65 ×10⁵-5.5 × 10⁵  Disposition of Toxic Drugs and Chemicals in(Acetylsalicylic acid) Man, 8th Edition, Biomedical Public, Foster City,CA, 2008, pp. 22-25 J Lab Clin Med. 1984 Jun; 103: 869 Ibuprofen 206.2924-32 1.16 × 10⁵-1.55 × 10⁵ BMC Clinical Pharmacology 2010, 10:10(Advil, Motrin) J Clin Pharmacol. 2001, 41: 330 Naproxen 230.26 Up to Upto J Clin Pharmacol. 2001, 41: 330 (Aleve) 60 2.6 × 10⁵

Example 3 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Mouse Bladder Smooth Muscle Cell Responses toInflammatory and Non-Inflammatory Stimuli Experimental Design

This study is designed to characterize how the optimal doses ofanalgesics determined in Example 2 affect bladder smooth muscle cells incell culture or tissue cultures, and to address whether differentclasses of analgesics can synergize to more efficiently inhibit COX2 andPGE2 responses.

The effectors, analgesic agents and antimuscarinic agents are describedin Example 2.

Primary culture of mouse bladder smooth muscle cells are subjected toshort term (1-2 hrs) or long term (24-48 hrs) stimulation with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

The cells are then analyzed for the release of PGH₂; PGE; PGE₂;Prostacydin; Thromboxane; IL-1β; IL-6; TNF-α; the COX2 activity; theproduction of cAMP and cGMP; the production of IL-1β, IL-6, TNF-α, andCOX2 mRNA; and surface expression of CD80, CD86, and WIC class IImolecules.

Materials and Methods Isolation and Purification of Mouse Bladder Cells

Bladder cells were removed from euthanized animals C57BL/6 mice (8-12weeks old), and cells were isolated by enzymatic digestion followed bypurification on a Percoll gradient. Briefly, bladders from 10 mice wereminced with scissors to fine slurry in 10 ml of digestion buffer (RPMI1640, 2% fetal bovine serum, 0.5 mg/ml collagenase, 30 μg/ml DNase).Bladder slurries were enzymatically digested for 30 minutes at 37° C.Undigested fragments were further dispersed through a cell-trainer. Thecell suspension was pelleted and added to a discontinue 20%, 40%. and75% Percoll gradient for purification on mononuclear cells. Eachexperiment used 50-60 bladders.

After washes in RPMI 1640, bladder cells were resuspended RPMI 1640supplemented with 10% fetal bovine serum, 15 mM HEPES, 2 mM L-glutamine,100 U/ml penicillin, and 100 μg/ml of streptomycin and seeded inclear-bottom black 96-well cell culture microculture plates at a celldensity of 3×10⁴ cells per well in 100 Cells were cultured at 37° C. ina 5% CO₂ atmosphere.

In Vitro Treatment of Cells with Analgesics

Bladder cells were treated with analgesic solutions (50 μl/well) eitheralone or together with carbachol (10-Molar, 50 μl/well), as an exampleof non-inflammatory stimuli, or lipopolysaccharide (LPS) of Salmonellatyphimurium (1 μg/ml, 50 μl/well), as an example of non-inflammatorystimuli. When no other effectors were added to the cells, 50 μl of RPMI1640 without fetal bovine serum were added to the wells to adjust thefinal volume to 200 μl.

After 24 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris, and stored at −70° C. for analysis of Prostaglandin E2 (PGE₂)responses by ELISA. Cells were fixed, permeabilized, and blocked fordetection of Cyclooxygenase-2 (COX2) using a fluorogenic substrate. Inselected experiment cells were stimulated 12 hours in vitro for analysisof COX2 responses

Analysis of COX2 Responses

COX2 responses were analyzed by a Cell-Based ELISA using Human/mousetotal COX2 immunoassay (R&D Systems), following the instructions of themanufacturer. Briefly, after cells fixation and permeabilization, amouse anti-total COX2 and a rabbit anti-total GAPDH were added to thewells of the clear-bottom black 96-well cell culture microcultureplates. After incubation and washes, an HRP-conjugated anti-mouse IgGand an AP-conjugated anti-rabbit IgG were added to the wells. Followinganother incubation and set of washes, the HRP- and AP-fluorogenicsubstrates were added. Finally, a Victor® V multilabel plate reader(PerkinElmer) was used to read the fluorescence emitted at 600 nm (COX2fluorescence) and 450 nm (GAPDH fluorescence). Results are expressed asrelative levels of total COX2 as determined by relative fluorescenceunit (RFUs) and normalized to the housekeeping protein GAPDH.

Analysis of PGE2 Responses

Prostaglandin E2 responses were analyzed by a sequential competitiveELISA (R&D Systems). More specifically, culture supernatants or PGE2standards were added to the wells of a 96-well polystyrene microplatecoated with a goat anti-mouse polyclonal antibody. After one hourincubation on a microplate shaker, an HRP-conjugated PGE2 was added andthe plates were incubated for an additional two hours at roomtemperature. The plates were then washed and HRP substrate solutionadded to each well. The color was allowed to develop for 30 minutes, andthe reaction stopped by the addition of sulfuric acid before reading theplate at 450 nm with wavelength correction at 570 nm. Results areexpressed as mean pg/ml of PGE2.

Other Assays

The release of PGH₂; PGE, Prostacydin; Thromboxane; IL-1β; IL-6; andTNF-α; the production of cAMP and cGMP; the production of IL-1β, IL-6,TNF-α, and COX2 mRNA; and surface expression of CD80, CD86, and MHCclass II molecules are determined as described in Example 2.

Results Analgesics Inhibit COX2 Responses of Mouse Bladder Cells to anInflammatory Stimulus

Several analgesics (acetaminophen, aspirin, ibuprofen, and naproxen)were tested on mouse bladder cells at the concentration of 5 μM or 50 μMto determine whether the analgesics could induce COX2 responses.Analysis of 24-hour cultures showed that none of the analgesics testedinduced COX2 responses in mouse bladder cells in vitro.

The effect of these analgesics on the COX2 responses of mouse bladdercells to carbachol or LPS stimulation in vitro was also tested. Asindicated in Table 1, the dose of carbachol tested has no significanteffect on COX2 levels in mouse bladder cells. On the other hand, LPSsignificantly increased total COX2 levels. Interestingly, acetaminophen,aspirin, ibuprofen, and naproxen could all suppress the effect of LPS onCOX2 levels. The suppressive effect of the analgesic was seen when thesedrugs were tested at either 5 μM or 50 μM (Table 4).

TABLE 4 COX2 expression by mouse bladder cells after in vitrostimulation and treatment with analgesic Total COX2 levels StimulusAnalgesic (Normalized RFUs) None None 158 ± 18 Carbachol (mM) None 149 ±21 LPS (1 μg/ml) None 420 ± 26 LPS (1 μg/ml) Acetaminophen (5 μM) 275 ±12 LPS (1 μg/ml) Aspirin (5 μM) 240 ± 17 LPS (1 μg/ml) Ibuprofen (5 μM))253 ± 32 LPS (1 μg/ml) Naproxen (5 μM) 284 ± 11 LPS (1 μg/ml)Acetaminophen (50 μM) 243 ± 15 LPS (1 μg/ml) Aspirin (50 μM) 258 ± 21LPS (1 μg/ml) Ibuprofen (50 μM) 266 ± 19 LPS (1 μg/ml) Naproxen (50 μM)279 ± 23

Analgesics Inhibit PGE2 Responses of Mouse Bladder Cells to anInflammatory Stimulus

The secretion of PGE2 in culture supernatants of mouse bladder cells wasmeasured to determine the biological significance of the alteration ofmouse bladder cell COX2 levels by analgesics. As shown in Table 5, PGE2was not detected in the culture supernatants of unstimulated bladdercells or bladder cells cultured in the presence of carbachol. Consistentwith COX2 responses described above, stimulation of mouse bladder cellswith LPS induced the secretion of high levels of PGE2. Addition of theanalgesics acetaminophen, aspirin, ibuprofen, and naproxen suppressedthe effect of LPS on PGE2 secretion, and no difference was seen betweenthe responses of cells treated with the 5 or 50 μM dose of analgesic.

TABLE 5 PGE2 secretion by mouse bladder cells after in vitro stimulationand treatment with analgesic. Stimulus Analgesic PGE2 levels (pg/ml)None None <20.5 Carbachol (mM) None <20.5 LPS (1 μg/ml) None 925 ± 55LPS (1 μg/ml) Acetaminophen (5 μM) 619 ± 32 LPS (1 μg/ml) Aspirin (5 μM)588 ± 21 LPS (1 μg/ml) Ibuprofen (5 μM)) 593 ± 46 LPS (1 μg/ml) Naproxen(5 μM) 597 ± 19 LPS (1 μg/ml) Acetaminophen (50 μM) 600 ± 45 LPS (1μg/ml) Aspirin (50 μM) 571 ± 53 LPS (1 μg/ml) Ibuprofen (50 μM) 568 ± 32LPS (1 μg/ml) Naproxen (50 μM) 588 ± 37

In summary, these data show that the analgesics alone at 5 μM or 50 μMdo not induce COX2 and PGE2 responses in mouse bladder cells. Theanalgesics at 5 μM or 50 however, significantly inhibit COX2 and PGE2responses of mouse bladder cells stimulated in vitro with LPS (1 μg/ml).No significant effect of analgesics was observed on COX2 and PGE2responses of mouse bladder cells stimulated with carbachol (1 mM).

Example 4 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Mouse Bladder Smooth Muscle Cell ContractionExperimental Design

Cultured mouse or rat bladder smooth muscle cells and mouse or ratbladder smooth muscle tissue are exposed to inflammatory stimuli andnon-inflammatory stimuli in the presence of analgesic agent and/orantimuscarinic agent at various concentrations. The stimulus-inducedmuscle contraction is measured to evaluate the inhibitory effect of theanalgesic agent and/or antimuscarinic agent.

The effectors, analgesic agents, and antimuscarinic agents are describedin Example 2.

Primary cultures of mouse bladder smooth muscle cells are subjected toshort term (1-2 hrs) or long term (24-48 hrs) stimulation with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

Materials and Methods

Primary mouse bladder cells are isolated as described in Example 3. Inselected experiments, cultures of bladder tissue are used. Bladdersmooth muscle cell contractions are recorded with a Grass polygraph(Quincy Mass., USA).

Example 5 Effect of Oral Analgesic Agents and Antimuscarinic Agents onCOX2 and Pge2 Responses of Mouse Bladder Smooth Muscle CellsExperimental Design:

Normal mice and mice with over active bladder syndrome are given oraldoses of aspirin, naproxen sodium, ibuprofen, Indocin, nabumetone,Tylenol, Celecoxib, oxybutynin, solifenacin, darifenacin, atropine, andcombinations thereof. Control groups include untreated normal mice anduntreated OAB mice with over active bladder syndrome. Thirty (30)minutes after last doses, the bladders are collected and stimulated exvivo with carbachol or acetylcholine. In selected experiments, thebladders are treated with botulinum neurotoxin A before stimulation withcarbachol. Animals are maintained in metabolic cages and frequency (andvolume) of urination are evaluated. Bladder outputs are determined bymonitoring water intake and cage litter weight. Serum PGH₂, PGE, PGE₂,Prostacydin, Thromboxane, IL-1β, IL-6, TNF-α, cAMP, and cGMP levels aredetermined by ELISA. CD80, CD86, and MHC class II expression in wholeblood cells are determined by flow cytometry.

At the end of the experiment, animals are euthanized, and ex vivobladder contractions are recorded with a Grass polygraph. Portions ofbladders are fixed in formalin, and COX2 responses are analyzed byimmunohistochemistry.

Example 6 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Human Bladder Smooth Muscle Cell Responses toInflammatory and Non-Inflammatory Stimuli Experimental Design

This study is designed to characterize how the optimal doses ofanalgesic determined in Examples 1-5 affect human bladder smooth musclecells in cell culture or tissue cultures and to address whetherdifferent classes of analgesics can synergize to more efficientlyinhibit COX2 and PGE2 responses.

The effectors, analgesic agents, and antimuscarinic agents are describedin Example 2.

Human bladder smooth muscle cells are subjected to short term (1-2 hrs)or long term (24-48 hrs) stimulation with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

The cells are then analyzed for the release of PGH₂; PGE; PGE₂;Prostacydin; Thromboxane; IL-1β; IL-6; TNF-α; the COX2 activity; theproduction of cAMP and cGMP; the production of IL-1β, IL-6, TNF-α, andCOX2 mRNA; and surface expression of CD80, CD86, and MHC class IImolecules.

Example 7 Effect of Analgesic Agents, Botulinum Neurotoxin andAntimuscarinic Agents on Human Bladder Smooth Muscle Cell ContractionExperimental Design

Cultured human bladder smooth muscle cells are exposed to inflammatorystimuli and non-inflammatory stimuli in the presence of an analgesicagent and/or antimuscarinic agent at various concentrations. Thestimuli-induced muscle contraction is measured to evaluate theinhibitory effect of the analgesic agent and/or antimuscarinic agent.

The effectors, analgesic agents, and antimuscarinic agents are describedin Example 2.

Human bladder smooth muscle cells are subjected to short term (1-2 hrs)or long term (24-48 hrs) stimulation with:

(1) Each analgesic agent alone at various doses.

(2) Each analgesic agent at various doses in the presence of LPS.

(3) Each analgesic agent at various doses in the presence of carbacholor acetylcholine.

(4) Each analgesic agent at various doses in the presence of AA, DGLA,or EPA.

(5) Botulinum neurotoxin A alone at various doses.

(6) Botulinum neurotoxin A at various doses in the presence of LPS.

(7) Botulinum neurotoxin A at various doses in the presence of carbacholor acetylcholine.

(8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA,or EPA.

(9) Each antimuscarinic agent alone at various doses.

(10) Each antimuscarinic agent at various doses in the presence of LPS.

(11) Each antimuscarinic agent at various doses in the presence ofcarbachol or acetylcholine.

(12) Each antimuscarinic agent at various doses in the presence of AA,DGLA, or EPA.

Bladder smooth muscle cell contractions are recorded with a Grasspolygraph (Quincy Mass., USA).

Example 8 Effect of Analgesic Agents on Normal Human Bladder SmoothMuscle Cell Responses to Inflammatory and Non Inflammatory SignalsExperimental Design Culture of Normal Human Bladder Smooth Muscle Cells

Normal human bladder smooth muscle cells were isolated by enzymaticdigestion from macroscopically normal pieces of human bladder. Cellswere expended in vitro by culture at 37° C. in a 5% CO₂ atmosphere inRPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES, 2 mML-glutamine, 100 U/ml penicillin, and 100 mg/ml of streptomycin andpassage once a week by treatment with trypsin to detach cells followedby reseeding in a new culture flask. The first week of culture, theculture medium was supplemented with 0.5 ng/ml epidermal growth factor,2 ng/ml fibroblast growth factor, and 5 μg/ml insulin.

Treatment of Normal Human Bladder Smooth Muscle Cells with Analgesics InVitro

Bladder smooth muscle cells trypsinized and seeded in microcultureplates at a cell density of 3×10⁴ cells per well in 100 μl were treatedwith analgesic solutions (50 μl/well) either alone or together carbachol(10-Molar, 50 μl/well), as an example of non-inflammatory stimuli, orlipopolysaccharide (LPS) of Salmonella typhimurium (1 μg/ml, 50μl/well), as an example of non-inflammatory stimuli. When no othereffectors were added to the cells, 50 μl of RPMI 1640 without fetalbovine serum were added to the wells to adjust the final volume to 200μl.

After 24 hours of culture, 150 μl of culture supernatants werecollected, spun down for 2 min at 8,000 rpm at 4° C. to remove cells anddebris, and stored at −70° C. for analysis of Prostaglandin E2 (PGE₂)responses by ELISA. Cells were fixed, permeabilized, and blocked fordetection of COX2 using a fluorogenic substrate. In selectedexperiments, cells were stimulated 12 hours in vitro for analysis ofCOX2, PGE2, and cytokine responses. Analysis of COX2, PGE2, and cytokineresponses

COX2 and PGE2 responses were analyzed as described in Example 3.Cytokine responses were analyzed as described in Example 2.

Results

Analgesics Inhibit COX2 Responses of Normal Human Bladder Smooth MuscleCells to Inflammatory and Non-Inflammatory Stimuli—

Analysis of cells and culture supernatants after 24 hours of culturesshowed that none of the analgesics tested alone induced COX2 responsesin normal human bladder smooth muscle cells. However, as summarized inTable 6, carbachol induced low, but significant COX2 responses in normalhuman bladder smooth muscle cells. On the other hand, LPS treatmentresulted in higher levels of COX2 responses in normal human bladdersmooth muscle cells. Acetaminophen, aspirin, ibuprofen, and naproxencould all suppress the effect of carbachol and LPS on COX2 levels. Thesuppressive effect of the analgesics was seen on LPS-induced responseswhen these drugs were tested at either 5 μM or

TABLE 6 COX2 expression by normal human bladder smooth muscle cellsafter in vitro stimulation with inflammatory and non-inflammatorystimuli and treatment with analgesic Total COX2 Total COX2 levels^(#)levels (Normalized (Normalized RFUs) RFUs) Stimulus Analgesic subject 1subject 2 None None 230 199 Carbachol 10⁻³M None (50 μM) 437 462Carbachol 10⁻³M Acetaminophen (50 μM) 298 310 Carbachol 10⁻³M Aspirin(50 μM) 312 297 Carbachol 10⁻³M Ibuprofen (50 μM) 309 330 Carbachol10⁻³M Naproxen (50 μM) 296 354 LPS (10 μg/ml) None 672 633 LPS (10μg/ml) Acetaminophen (5 μM) 428 457 LPS (10 μg/ml) Aspirin (5 μM) 472491 LPS (10 μg/ml) Ibuprofen (5 μM) 417 456 LPS (10 μg/ml) Naproxen (5μM) 458 501 LPS (10 μg/ml) Acetaminophen (50 μM) 399 509 LPS (10 μg/ml)Aspirin (50 μM) 413 484 LPS (10 μg/ml) Ibuprofen (50 μM) 427 466 LPS (10μg/ml) Naproxen (50 μM) 409 458 ^(#)Data are expressed as mean ofduplicates

Analgesics Inhibit PGE2 Responses of Normal Human Bladder Smooth MuscleCells to Inflammatory and Non-Inflammatory Stimuli—

Consistent with the induction of COX2 responses described above, bothcarbachol and LPS induced production of PGE2 by normal human bladdersmooth muscle cells. Acetaminophen, aspirin, ibuprofen, and naproxenwere also found to suppress the LPS-induced PGE2 responses at either 5μM or 50 μM (Table 7).

TABLE 7 PGE2 secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non-inflammatory stimuli andtreatment with analgesic PGE2 PGE2 levels levels^(#) (pg/ml) (pg/ml)Stimulus Analgesic Subject 1 Subject 2 None None <20.5 <20.5 Carbachol10⁻³M None 129 104 Carbachol 10⁻³M Acetaminophen (50 μM) 76 62 Carbachol10⁻³M Aspirin (50 μM) 89 59 Carbachol 10⁻³M Ibuprofen (50 μM) 84 73Carbachol 10⁻³M Naproxen (50 μM) 77 66 LPS (10 μg/ml) None 1125 998 LPS(10 μg/ml) Acetaminophen (5 μM) 817 542 LPS (10 μg/ml) Aspirin (5 μM)838 598 LPS (10 μg/ml) Ibuprofen (5 μM) 824 527 LPS (10 μg/ml) Naproxen(5 μM) 859 506 LPS (10 μg/ml) Acetaminophen (50 μM) 803 540 LPS (10μg/ml) Aspirin (50 μM) 812 534 LPS (10 μg/ml) Ibuprofen (50 μM) 821 501LPS (10 μg/ml) Naproxen (50 μM) 819 523 ^(#)Data are expressed as meanof duplicates

Analgesics Inhibit Cytokine Responses of Normal Human Bladder Cells toInflammatory Stimuli—

Analysis of cells and culture supernatants after 24 hours of cultureshowed that none of the analgesics tested alone induced IL-6 or TNFαsecretion in normal human bladder smooth muscle cells. As shown inTables 8 and 9, the doses of carbachol tested induced low, butsignificant TNFα and IL-6 responses in normal human bladder smoothmuscle cells. On the other hand, LPS treatment resulted in massiveinduction of these proinflammatory cytokines. Acetaminophen, aspirin,ibuprofen, and naproxen suppress the effect of carbachol and LPS on TNFαand IL-6 responses. The suppressive effect of the analgesics onLPS-induced responses was seen when these drugs were tested at either 5μM or 50 μM.

TABLE 8 TNFα secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non-inflammatory stimuli andtreatment with analgesic TNFα TNFα (pg/ml)^(#) (pg/ml) Stimuli AnalgesicSubject 1 Subject 2 None None <5 <5 Carbachol 10⁻³M None 350 286Carbachol 10⁻³M Acetaminophen (50 μM) 138 164 Carbachol 10⁻³M Aspirin(50 μM) 110 142 Carbachol 10⁻³M Ibuprofen (50 μM) 146 121 Carbachol10⁻³M Naproxen (50 μM) 129 137 LPS (10 μg/ml) None 5725 4107 LPS (10μg/ml) Acetaminophen (5 μM) 2338 2267 LPS (10 μg/ml) Aspirin (5 μM) 24792187 LPS (10 μg/ml) Ibuprofen (5 μM) 2733 2288 LPS (10 μg/ml) Naproxen(5 μM) 2591 2215 LPS (10 μg/ml) Acetaminophen (50 μM) 2184 2056 LPS (10μg/ml) Aspirin (50 μM) 2266 2089 LPS (10 μg/ml) Ibuprofen (50 μM) 26031997 LPS (10 μg/ml) Naproxen (50 μM) 2427 2192 ^(#)Data are expressed asmean of duplicates.

TABLE 9 IL-6 secretion by normal human bladder smooth muscle cells afterin vitro stimulation with inflammatory and non-inflammatory stimuli andtreatment with analgesic IL-6 IL-6 (pg/ml)^(#) (pg/ml) StimulusAnalgesic Subject 1 Subject 2 None None <5 <5 Carbachol 10⁻³M None 232278 Carbachol 10⁻³M Acetaminophen (50 μM) 119 135 Carbachol 10⁻³MAspirin (50 μM) 95 146 Carbachol 10⁻³M Ibuprofen (50 μM) 107 118Carbachol 10⁻³M Naproxen (50 μM) 114 127 LPS (10 μg/ml) None 4838 4383LPS (10 μg/ml) Acetaminophen (5 μM) 2012 2308 LPS (10 μg/ml) Aspirin (5μM) 2199 2089 LPS (10 μg/ml) Ibuprofen (5 μM) 2063 2173 LPS (10 μg/ml)Naproxen (5 μM) 2077 2229 LPS (10 μg/ml) Acetaminophen (50 μM) 2018 1983LPS (10 μg/ml) Aspirin (50 μM) 1987 2010 LPS (10 μg/ml) Ibuprofen (50μM) 2021 1991 LPS (10 μg/ml) Naproxen (50 μM) 2102 2028 ^(#)Data areexpressed as mean of duplicates

Primary normal human bladder smooth muscle cells were isolated, culturedand evaluated for their responses to analgesics in the presence ofnon-inflammatory (carbachol) and inflammatory (LPS) stimuli. The goal ofthis study was to determine whether or not normal human bladder smoothmuscle cells recapitulate the observations previously made with murinebladder cells.

The above-described experiment will be repeated with analgesic agentsand/or antimuscarinic agents in delayed-release, or extended-releaseformulation or delayed-and-extended-release formulations.

The above description is for the purpose of teaching a person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1. A method for reducing frequency of urination in a subject,comprising: administering to a subject in need thereof a pharmaceuticalcomposition comprising an effective amount of one or more analgesicagents and an effective amount of tadalafil, wherein said pharmaceuticalcomposition is formulated for immediate release or delayed release orextended release.
 2. The method of claim 1, wherein said pharmaceuticalcomposition is formulated for immediate release.
 3. The method of claim1, wherein said pharmaceutical composition is formulated for extendedrelease.
 4. A method for reducing frequency of urination in a subject,comprising: administering to a subject in need thereof a pharmaceuticalcomposition comprising: an active ingredient comprising one or moreanalgesic agents in an amount of 1-2000 mg per agent; and an effectiveamount of an inhibitor of phosphodiesterase type 5 (PDE, 5 inhibitor),wherein said one or more analgesic agents are selected from the groupconsisting of aspirin, ibuprofen, naproxen, naproxen sodium;indomethacin, nabumetone, and acetaminophen, wherein said pharmaceuticalcomposition is formulated for immediate release or delayed release orextended release.
 5. The method of claim 4, wherein said pharmaceuticalcomposition is formulated for delayed release.
 6. The method of claim 4,wherein said pharmaceutical composition is formulated for extendedrelease.
 7. The method of claim 6, wherein said pharmaceuticalcomposition is formulated for immediate release. 8-10. (canceled)
 11. Amethod for reducing the frequency of urination in a subject, comprising:administering to a subject in need thereof a pharmaceutical compositioncomprising: an effective amount of a first active ingredient comprisingone or more analgesic agents and tadalafil; and an effective amount of asecond active ingredient comprising one or more agents selected from thegroup consisting of analgesic agents, antimuscarinic agents,antidiuretics, spasmolytics, PDE 5 inhibitors and zolpidem, wherein saidpharmaceutical composition is formulated for immediate release ordelayed release or extended release.
 12. The method of claim 11, whereinsaid pharmaceutical composition is further coated with an entericcoating.
 13. The method of claim 11, wherein said first activeingredient comprises acetaminophen.
 14. The method of claim 11, whereinsaid first active ingredient further comprises an antimuscarinic agent,an antidiuretic, a spasmolytic or zolpidem. 15-20. (canceled)
 21. Themethod of claim 1, wherein said pharmaceutical composition is formulatedfor delayed release.
 22. The method of claim 11, wherein saidpharmaceutical composition is formulated for immediate release.
 23. Themethod of claim 11, wherein said pharmaceutical composition isformulated for delayed release.
 24. The method of claim 11, wherein saidpharmaceutical composition is formulated for extended release.
 25. Amethod for reducing frequency of urination in a subject, comprising:administering to a subject in need thereof a pharmaceutical compositioncomprising an effective amount of one or more analgesic agents and aneffective amount of a PDE 5 inhibitor, wherein the pharmaceuticalcomposition comprises an immediate-release component and an extendedrelease component.
 26. The method of claim 25, wherein theimmediate-release component comprises the one or more analgesic agents,and wherein the extended release component comprises the PDE 5inhibitor.
 27. The method of claim 25, wherein the one or more analgesicagents comprises acetaminophen.
 28. The method of claim 27, wherein theone or more analgesic agents comprises acetaminophen and a non-steroidalanti-inflammatory drug.
 29. The method of claim 25, wherein the PDE 5inhibitor is tadalafil.